Spectroscopic analysis of genetically modified photosynthetic reaction centers.

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Annu. Rev. Biophys. Biop/tys. Chern. 1990. 19:333-67 Copyright © 1990 by Annual Reviews Inc. All rif{hts reserved

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SPECTROSCOPIC ANALYSIS OF GENETICALLY MODIFIED PHOTOSYNTHETIC REACTION CENTERS William J. Coleman and Douglas C. Youvan

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139 KEY WORDS:

electron

transfer,

membrane

chemistry, Rhvdobacter.

proteins,

mutagenesis,

photo

CONTENTS PERSPECTIVES AND OVERVIEW

............................. . ...................................... . . . . . . ..............

A GENETIC SYSTEM FOR MUTAGENESIS

. . . . .............. . ................................... . . . . .............. . . . . .

PIGMENT BINDING AND THE CHARGE TRANSFER PROPERTIES OF THE PRIMARY ELECTRON DONOR

333 336 340

....................................................................... . . . . . . . . ............ . . . . . . . . . .... .

REPLACEMENT OF THE AXIAL LIGANDS TO THE MONOMERIC BACTERIOCHLOROPHYLLS

......

349

THE SPECTROSCOPIC RED SHIFT OF THE PHOTOACTIVE BACTERIOPHEOPHYTIN ............ . . . . . . .

351

. ......................... Electron Transfer to the Primary Quinone: QA Binding and Reduction .. ................... Mutations Affecting Q8 and Herbicide Binding ......... . . . . . . .......... ................................

354 354 356

THE QUINONE ELECTRON ACCEPTORS

................... ............. . . . . ....... . . . . .....

MUTATIONS INVOLVING OTHER COMPONENTS OF THE PHOTOSYNTHETIC APPARATUS

.........

Electron Donation./i"om Cytochrome C2 to the Oxidized Special Pair ........................ Bacteriochlorophyll Binding in the Light-Harvesting I Antenna ...... .......................... CONCLUDING REMARKS

.......

... ... .... ...................................... ..........................................

359 359

360

362

PERSPECTIVES AND OVERVIEW

The photosynthetic reaction center (RC) from the purple bacteria is a membrane-bound pigment-protein complex containing four bacterio chlorophylls (Bchl), two bacteriopheophytins (Bphe), two quinones (Q), and a ferrous nonheme iron atom (Fe) (5 1 , 6 1 , 80, 1 1 7). These com333 0883-9 1 82/90/06 1 0-033 3$02.00


334

COLEMAN & YOUVAN

ponents are attached to two symmetrically arrangcd protein subunits (L and M), which along with a third subunit (H), comprise the basic RC core structure. The L and M subunits each contain five transmembrane a-helices (designated A through E) and two short, connecting helices (design ated cd and de). Together, the L and M subunits form a dimeric complex in which the chromophores are arranged in pairs around an approximate C 2 axis of symmetry (Figure 1) (2, 1 1 6, I 1 7) . The axis runs from the special pair dimer of Bchls on the periplasmic side of the RC to the Fe on the cyto plasmic side. Excitation cncrgy is transferred into the RC complex from the surrounding light-harvesting (LH) pigment-protein complexes ( 1 8, 55, 192), whose three-dimensional structures are not yet known. Despite the overall structural symmetry of the RC, electron transfer within the system is asymmetric [for a discussion of RC electron transfer, see Boxer's review in this volume (9a) and 22, 65, 88, 1 20, 1 3 1J. The initial

Figure I

X-ray crystallographic structure of the photochemical core of the RC from

Rhodopseudomonas viridis. All nine of the RC prosthetic groups are displayed in the upper

stereo pair. The backbone of the RC, L, and M subunits has been added to the lower stereo pair. Two other polypeptide chains, the H subunit and a cytochrome, have been omitted for graphic clarity. The prosthetic groups are labeled according to a convention that "A-branch" prosthetic groups lie on the same side of the RC as the primary quinone (QA) and that "B branch" prosthctic groups lic on the same side as the secondary quinone (Qu). The prosthetic groups are abbreviated: P, special pair bacteriochlorophyll; B, monomeric bacterio chlorophyll; H, bacteriopheophytin; Q, quinone; Fe, ferrous iron ion.


GENETICALLY MODIFIED REACTION CENTERS

335

photochemical event leading to charge separation results in the oxidation of P (the Bchl dimer, also known as the special pair) to P+ and the reduction of Bphe (known as H) to H-. Although two potential electron transfer pathways lie within the RC (designated here as A and B), spec troscopic evidence indicates that only HA is reduced following an excitation flash ( 1 4, 26, 94, 97, 98). Investigators actively seek an answer to the question of whether the monomeric (accessory) Bchl on the A-branch (known as BA) participates in this process (64, 79). The reduced acceptor H;;: subsequently transfers an electron to the primary quinone (QA), a one electron acceptor ( 1 25). Two charge separations allow Q;;: to reduce the secondary quinone (QB) to form QBH2 ( 1 80), which then dissociates from its binding site and passes its reducing equivalents to the cytochrome bCI oxidoreductase complex (43, 45). The cyclic electron transfer pathway, whose energy is ultimately used to generate ATP (44, 1 27), is completed when a high-potential cytochrome reduces p+ (57, 1 29, 1 38, 1 57). Explaining the physicochemical basis for the rapidity and directionality of electron transfer in the RC has been one of the primary goals of structure-function studies. Before the DNA sequence of the RC structural genes (54, 1 1 8, 1 1 9, 1 70, 1 72, 1 73, 1 87) and the three-dimensional structure of the protein complex (2, 3, 33, 50, 1 1 7) had been elucidated, investigations of RC function focused predominantly on the spectroscopic properties (e.g. the electronic structure) of the chromophores and the interactions between them ( 1 25). However, these properties clearly depended, in many cases, on unknown interactions between the prosthetic groups and the protein itself ( 1 9, 63, 1 23, 1 79). The RC protein may perform a number of important roles, including: (a ) providing a scaffold for the prosthetic groups by fixing the orientations of the chromophores and the distances between them; (b) promoting stable charge separation by adjusting the energy levels/electron distributions within the donor and acceptor states; or (c) facilitating clectron transfcr by providing pathways for forward electron movement or by impeding wasteful charge recombination reac tions. With the advent of the high-resolution X-ray crystal structures, defining the protein ligands responsible for chromophore and metal bind ing and identifying amino acid side chains along the two chromophore pathways that break the symmetry of the protein became possible ( 1 5 6, 1 82). Figures 1 , 4, 6, 7 and 9 highlight many of the important structural features of the Rhodopseudomonas viridis RC. This structure has been refined to 2.3-A resolution (5 1 ) and atomic coordinates have recently been deposited in the Brookhaven Protein Data Bank. In vitro mutagenesis methods have now made it relatively easy to intro duce specific alterations in the protein structure, so that hypotheses regard ing the role of the protein in assembling the photosynthetic complexes,


336

COLEMAN & YOUVAN

binding the prosthetic groups, and modifying the spectroscopic and elec tron transfer properties of the RC can be tested (32, 190). In addition, protein engineering of the bacterial RC has generated a potentially useful model system for studying herbicide binding and for gaining insight into the molecular architecture of the plant Photosystem II (PS II), whose three-dimensional structure is not yet known (see Refs. 25, 4 1 , 68, 1 1 6, 122, 159, 164, 187). It may also be possible in the future to manipulate this system to examine the folding of membrane proteins since thc availability of spectrally distinct "reporter groups" throughout the structure makes it feasible to screen complex libraries of mutations for isomorphic amino acid sequences. The goal of this review is to summarize the progress to date in the construction and characterization of genetically modified RCs and LH complexes. Directed mutations have been introduced in the vicinity of each of the different prosthetic groups in the Rhodobacter capsulatus RC, extending from the special pair to the QB pocket. Several mutations have also been made at critical residues in the IX subunit of LH I, one of the two antenna pigment complexes in this organism. Since at the time of writing, this is the only purple bacterial system for which engineered photosynthetic mutants have been published, the discussion of spectro scopic characterization of modified RCs is mostly limited to the Rb. capsulatus system. Recently, however, partial deletion backgrounds for Rb. sphaeroides have been constructed, which will make possible the introduction of directed mutations in this system. In plants, mutations directed at the electron-donating tyro sines in the PS II RCs have already been constructed and characterized in cyanobacteria (48, 163). A number of spontaneous, herbicide-resistant mutants from Rps. viridis and Rh. sphaeroides have also been isolated, and we discuss these in the section devoted to QB. A GENETIC SYSTEM FOR MUTAGENESIS

Design requirements for an optimal system to mutagenize the photo synthetic apparatus of the purple bacteria (32) include the following factors: 1 . The organism must be capable of expressing the photosynthetic genes while growing nonphotosynthetically. The availability of alternative growth modes allows researchers to culture photosynthetically impaired mutants without selecting for unwanted revertants, since the latter contain primary and/or secondary mutations. 2. The structural genes encoding the proteins to be m utagenized must be completely deleted from the chro mosome. This type of deletion background makes regeneration of arti factual wild-type revertants by homologous recombination between a par-



337

tial chromosomal gene and the mutant plasmid gene impossible. 3. A retrievable plasmid vector (containing the appropriate operon and a muta genized copy of the gene) should be used to complement the deletion background in trans. Plasmid retrieval enables one to check the correctness of the engineered mutation and/or to characterize spontaneous mutations in the operon, while avoiding the more lengthy procedure of cloning mutated genes from the bacterial chromosome. Many members of the family Rhodospirillaceae express the photo synthetic apparatus in the light in response to lowered oxygen tension ( I , 6, 7, 56, 1 47). However, Rh. capsulatus, Rh. sphaeroides, and some other species also express the photosynthetic apparatus during semiaerobic growth in the dark (112, 184, 186). This adaptability makes the main tenance of RC deletion backgrounds by means of nonphotosynthetic growth modes possible (186). Microaerophilic culture of photo synthetically defective Rh. capsulatus mutants has been routinely used to obtain large quantities of modified RCs for certain types of biophysical analysis (30, 139). Rh. capsulatus contains a low-level constitutive promoter within the puf Q gene that enables this organism to continue to express the photosynthetic apparatus during the transition from aerobiosis to anaerobic photosynthetic growth (185). Deletion backgrounds have now been constructed for both the Rh. cap sulatus (189) and Rh. sphaeroides (47, 60) puf operon. The puf operon contains the pufQ gene (6), the LH I r:t. and f3 subunit genes (85, 187), the L and M RC subunit genes (172, 173, 187), and the pufX gene (96, 187). Deletion of the Rh. sphaeroides puh A operon [containing the structural gene for the RC H-subunit ( lS I , 187)] has also been achieved (151). A deletion background for the puc operon [containing the genes for the LH II/B800-850 complex ( 5 , 86, 188)] has been constructed in Rh. capsulatus (189). By using interposon mutagenesis (146), researchers have developed strains of Rb. capsulatus that lack the L, M , and LH I structural genes (189). In this case, the Rh. capsu!atus gene-transfer agent (183) was used to generate a background in which the genes for LH I r:t. and /3, L, M, and Puf X have been completely deleted and replaced by a spectinomycin interposon (i.e. an insertion-deletion). Use of this method also generated the Rh. capsulatus deletion background strain U43, which lacks the RC complex and both LH antennae (189). A plasmid vector containing either the wild-type or a mutagenized copy of the puf operon can complement the U43 deletion background. Hence, only the copy of the puf operon encoded by the plasmid is capable of being expressed. Rh. capsulatus puf deletions have also been constructed using plasmid-mediated recom binational techniques (34). A partial deletion mutant has recently been developed for Rb. sphaero-

338

COLEMAN & YOUVAN

ides that has the puf X and puf M genes completely deleted, along with

60% of the puf L gene (corresponding to amino acid residues 1 1 5-28 1 ) (60). I n this construction, the deleted genes were replaced by a kanamycin


interposon. Prior to this work, a puf- strain of Rb. sphaeroides had been made by deleting a small portion of the 5 ' end of the put' operon and replacing it with a kanamycin-resistance gene (47). A pull A deletion mutant containing an inserted kanamycin cartridge has also been con structed in Rb. sphaeroides ( 1 5 1 ) . Progress i n developing complementing plasmids for Rb. capsulatus mutagenesis was greatly assisted by the construction of the vector pU2922 (25). Plasmid pU2922 is a fusion of a broad host-range conjugative plasmid (pRK292) and a previously engineered pBR322 derivative [pU22 (24)] carrying the puj operon. The new plasmid is a signi ficant improvement over pBR derivatives since it can be conjugated at very high frequency from E. coli to Rhodobacter. Plasmid pU2922 is incorporated i nto the current Rh. capsulatus mutagenesis procedure through the following steps (Figure 2): 1. Site-directed mutations are constructed in M l 3 mp1 8 or

-

Q

EJl..nH

Q 1.

H

K

1

,

U15g

Q

2

�s

put, crtD

3

"�'�1' ""7 0 Modified pU29

E

S

Modified

QB

K

M

Q B X

� �s PU 9

�

Subclone.

E

S

pU2922

S

M13

0

)

pU2922

4

X

U43b put, puc, crt

5

-

0 E

S

Modified

pU2922

Mutant

517

Rb. capsulatus

Ira E. coli

•

6117

Fi,qure 2 An extensive system of bacterial strains and plasmids has been developed to manipulate the photosynthetic apparatus genes. Genetically engineered plasmids can be constructed in E. coli strains and then conj ugated into Rhodobacter capsulatus deletion backgrounds. Each numbered step in the figure is described in the text. An EcoRI-HindIII fragment carries the LH I f3 and ex genes (pur B and pur A); the HindIII-KpnI fragment carries the L subunit (purL); the Kpnl-BamHI fragment carries the M subunit (purM); the B m HI SacI fragment carries pufX. Plasmid pU2922 can be reconstructed using a unique EcoRI-Sac I fragment that carries all of the structural genes and then conjugated from E. coli strain S17-1 to various Rhodobacter deletion backgrounds. Thc three deletion back grounds shown in this figure possess different point mutations that modify carotenoid expression and/or inactivate LH II genes. A similar system of plasmids and deletion back grounds has been developed for the puc operon, which expresses the LH II structural genes.

u -



339

mp l 9 subclones of the puf operon using Kunkel's procedure ( 1 00); 2. A plasmid pU29 derivative bearing the site-directed mutation is recon structed from wild-type pU29 and the modified replicative form (RF) of M 1 3; 3. The reconstructed puf operon is shuttled from pU29 to pU2922 via a 3500-bp Eca RI-Sac I fragment; 4. pU2922 is conjugated from a tra+ strain of E. cali [S1 7-1 ( 1 48)] into an Rb. capsulatus puf deletion back ground (e.g. U43); 5. The resulting transconjugant (in this case, a mutant) is selected and characterized. Ground-state absorption spectroscopy on chromatophore membranes can be used to demonstrate the expression of RC and LH genes in a deletion background (Figure 3).

z

o

6:a:

o (f) (Q « w >

5w

a: 500 Figure 3

A 600 700 800 WAVELENGTH (nm)

900

Ground-state absorption spectra of chromatophore membranes from two different

Rh. capsula/us mutants. These spectra illustrate the use of chromophoric signals to assess

RC and LH phenotypes. Absorbance at 880 nm primarily results from LH I, while lesser absorbances at 760 nm and 800 nm primarily result from the RC. While Bchl Qy transitions are red-shifted to different extents in the near-infrared, all Bchls, regardless of their environ ments, have a Qx transition at about 590 nm. (Spectrum A) Deletion strain U43 is devoid of all absorbances characteristic of LH and RCs. The gradual increase in absorption at shorter wavelengths results from light scattering by the membrane. (Spectrum B) RC and LH I expression is restored by returning plasmid pU2922 to the U43 deletion background.


340

COLEMAN & YOUVAN

This system has the added feature that the integrity of any engineered mutation in Rhodobacter can be rechecked by extracting the plasmid (using an alkaline-SDS miniprep), subcloning (Figure 2, step 7), and sequencing the mutagenized region. One may also select for spontaneous mutants or revertants bearing primary or second-site mutations on this plasmid while the plasmid is maintained in a deletion background, i.e. [U43(pU2922»). This procedure has an advantage over cloning mutated genes from the bacterial chromosome in that there is about 1 03 less DNA to screen. In more complex schemes, further selection against the phenotype of the modified plasmid may generate new mutant backgrounds, which can be subsequently cured of plasmids by relaxing the antibiotic selection for the plasmid over several generations (Figure 2, step 6). The procedures described here were used to generate the mutant Rb. capsulatus phenotypes discussed in the next sections.

PIGMENT BINDING AND THE CHARGE TRANSFER PROPERTIES OF THE PRIMARY ELECTRON DONOR

The primary charge separation event in the RC involves capture of a photon by the special pair Bchl dimer to generate P*, whose lowest (n,n*) singlet excited state donates an electron to HA (over a center-to-center distance of approximately 17 A) in �3-4 ps at room temperature ( 17, 89, 1 13, 174). A fundamental question related to the dynamics of charge separation concerns whether the p* excited state contains some intradimer charge-transfer (CT) character (i.e. [BchltBchIB) or [BchlABchltD (for discussion, see Refs. 65, 74, 1 33 , 145). In addition, researchers have con siderable interest in determining how such a state might form during the initial exitation event (75, 1 44, 1 68, 176), since calculations on wild-type RCs suggest that the CT states of the primary donor lie above p* in energy ( 132, 133). Results of Stark-effect spectroscopy on wild-type Rb. sphaeroides RCs ( 102-1 04) and hole-burning experiments on Rb. sphaero ides and Rps. viridis RCs ( 1 1, 1 2) have led to the proposal that photo excitation develops directly into a state that has a large dipole moment. However, the observation that only very broad holes (�400 cm- I ) can be burned into the long-wavelength absorption band of the dimer in both Rb. sphaeroides and Rps. viridis RCs, corresponding to a P* lifetime considerably shorter than 3-4 ps ( 1 1 4, 1 1 5), has been interpreted to mean that the initial neutral excited state evolves on an ultrafast timescale (�25 fs) into a charge-separated state of the Bchl dimer (see discussion in Ref. 1 7 1) .



341

In the wild-type RC, the two Bchls that comprise the dimer are related by C2 symmetry (Figure 4), and thus in the absence of solvent effects, any potential charge-transfer state ought to be shared between the two possible configurations. The X-ray crystal structures of both Rps. viridis and Rb. sphaeroides, however, reveal substantial asymmetry in the protein environment surrounding the dimer, particularly with respect to the arrangement of hydrogen bonds between the Bchl ring substituents and the protein (22, 82, 1 35, 1 82). These interactions may be responsible for the observed inequality in the spin distribution of the triplet ep) in EPR measurements on single crystals of Rps. viridis RCs ( 124). These data led to the conclusion that the triplet, which forms from the recombination of the state P+ HA , resides mainly on the A-branch Bchl of the dimer (denoted here as BchlA or PA)' Absorbance-detected magnetic resonance (ADMR) spectroscopy also indicates triplet localization on the BchlA of the dimer ( 1 07). The triplet in Rh. sphaeroides, however, is more symmetrically distributed ( 1 24). EPR measurements of the Iinewidth of the P+ radical cation support the idea of an unequal sharing of the unpaired spin density in Rps. viridis ( 1 24).

Figure 4

This stereo pair illustrates the interaction of thc Land M subunit D-helices with

the special pair Bchls in Rps. viridis. The ligands to the central Mg atom of the special pair Bchls are the side chains of histidines L-I73 and M -200 (coordinated to

PA

and

P B, respec

tively). This view is rotated about the pseudo-C2 symmetry axis, in relation to Figure I. such that the Bchl rings are displayed nearly edge-on.


342

COLEMAN & YOUVAN

The special pair is located on the periplasmic end of the RC (Figure 1, bottom face of the structure), where the A-branch lies predominantly within the L-subunit and the B-branch lies predominantly within the M subunit. However, on the cytoplasmic end of the RC, two of the trans membrane a-helices (D and E) have crossed over to the other side. Thus, the stereo pairs reveal that the L-subunit D-helix interacts with PA and QB, while the symmetrically disposed M-subunit D-helix interacts with PB and QA. While an X-ray structure is not currently available for Rb. capsulatus, the 3-dimensional structure of the L- and M-subunits is expected to be very similar to the Rps. viridis structure, as indicated by similar hydropathy plots and extensive sequence identity ( 1 1 6, 1 1 8, 1 87). One can make maximum homology sequence alignments between the two L-subunits and between the two M-subunits of these species without introducing any gaps; therefore, homologous residues have identical residue numbers. Within the dimer itself, replacement of one of the Bchls with a Bphe serves as a means to artificially disrupt the symmetry of the two Bchls. In Rb. capsulatus, site-directed mutagenesis of histidine M-200 on the D helix, the ligand to the Bchl on the B-branch (denoted here as BchlB or PB), results in a Rchl

--->

Rphe replacement when His is mutagenized to Leu

2 or Phe (30). Measurement of the pigment composition of HisM 00 ---> Leu RCs indicates a total tetrapyrrole pigment content of 5.7 ± 0. 1 and a Bchl/Bphe ratio of 1 .0 ± 0. 1 . The HisM200 ---> Phe RCs have a total pigment content of 5.8 ± 0.2 and a Bchl/Bphe ratio of 1 . 1 ± 0. 1 . Replacing the axial His ligand with GIn at either M-200 or the equivalent position on the L-side (L- I 73) did not result in a significant change in the pigment composition of the RCs. Cultures of Rb. capsulatus possessing the latter two mutations are capable of growing photosynthetically on plate assays, whereas both the HisM200 ---> Leu and HisM200 ---> Phe cells are incapable of photosynthetic growth (30). The most noticeable difference between the room temperature ground state absorption spectra of wild-type and mutant RCs occurs in the HisM200 ---> Leu and HisM200 ---> Phe RCs, in which the 850-nm absorption peak attributable to the lowest-energy singlet transition of the dimer (the Qy band) is replaced by a broad, diffuse band (Figure 5) (30). In the HisM200 ---> Leu RCs, the absorption between 900 and 950 nm and between 750 and 800 nm is actually somewhat greater than in wild-type, but the overall oscillator strength between 700 and 9 50 nm is slightly reduced (91 ) . A t the positions corresponding t o the Q x bands o f the Bphe and Bchl (ca. 530-6 1 0 nm), the Bphe amplitude increases and the Bchl amplitude decreases, relative to the wild-type spectrum in both the HisM200 ---> Leu and HisM2uU ---> Phe RCs. In combination with the pigment-extraction data, these results indicate that each of these mutations produces a [Bchl-Bphe]


343

z

o


I0... a:

o (f) ro « w >

�w a:

400 Figure 5

500

800 600 700 WAVELENGTH (nm)

900

Ground-state absorption spectra of genetically altered RCs wherein the axial histidine ligands to the special pair Bchls have been separately replaced with leucines. Both spectra are very similar to wild-type (see Figure 1 0, spectrum A) with the important excep tion o[the long wavelength absorption band assigned to the special pair. Both modified RCs show a significant broadening of this band, which, along with other evidence (e.g. pigment extractions), indicates that the special pair Bchl dimer has been replaced by a [Bchl-Bphe] heterodimer. Broadening of the 850-nm band may result from the formation of an intradimer charge-transfer state.

heterodimer at the special pair (30, 9 1 ). This assignment is consistent with the reduction of oscillator strength in the near-infrared and with the blue shift in the long-wavelength absorption band for the dimer, since one might expect insertion of a Bphe to affect the excitonic coupling and/or the separation and geometry between the two halves of the dimer, as well as the charge-transfer character of the dimer excited state (9 1 ). Oxidized-min us-reduced (light-min us-dark) difference spectra at room temperature for the single mutants (His Ll73 � Gin and HisM200 � GIn) and for the double mutant (HisLl73,M200 � GIn) are essentially the same as in wild-type, as are the measured rates (kr) for the recombination reaction P+QA" � PQA (30). The heterodimer Res can also perform charge sep aration, but their difference spectra are significantly changed from wild-


344

COLEMAN & YOUVAN

type. Although one observes the putative electrochromic shift on the 800nm absorption peak of the monomeric Bchls, the Qx component of the special pair at approximately 600 nm is not bleached. In the Qy region, the absorption change at 850 nm due to bleaching of the special pair is less than that of wild-type because of the lower oscillator strength at this wavelength in the heterodimer RCs. The rate of the recombination reaction (kr) is also faster (30). Researchers have investigated the electron transfer properties of the HisM200 ---+ Leu heterodimer mutant in greater detail using subpicosecond time-resolved optical measurements. Comparison with wild-type Rh. cap sulatus RCs (89, 9 1 ) supports the finding that the HisM200 ---+ Leu RCs are capable of generating a charge-separated state (D+HA), where D is used instead ofP to denote the primary donor in the heterodimer RCs. However, the overall yield is only �50% of that obtained for wild-type RCs con taining a [Bchlb homodimer (9 1 ). In wild-type Rh. capsulatus, the time constant for the decay of P*HA to P+HA' based on the decay of stimulated emission, is 3.5 ±0.6 ps (89, 91). This reaction occurs with essentially 1 00% yield. But in the HisM200 ---+ Leu RCs, the lifetime of D* increases to 14 ± 3 p s and decays via forward electron transfer to D+HA, at an estimated rate of (�25 pS)-1 and via return to the ground state at an estimated rate of (�30 pS)- 1 (9 1 ) . Electron transfer from HA to QA occurs with a 1 00% yield, as in wild-type, but the rate actually speeds up slightly from (21 0 pS)- 1 in wild-type to ( l IS pS)- 1 in the HisM200 ---+ Leu RCs at room tem perature (9 1 ). Rapid decay of D* t o the ground state and slow electron transfer to HA are consistent with the possibility that excitation of D produces a partial intradimer CT state [Bchl+ Bphe-] (87). Depending on the nature of the surrounding medium, mixing between a CT state and D* in the het erodimer is expected to occur more readily than mixing between a CT state and p* in the wild-type homodimer (e.g. 74, 1 34) because [Bchl+ Bphe-] is predicted to lie at lower energy than [Bchl+Bchl-]. Rapid deactivation to the ground state may then reflect charge recombination within a het erodimer radical pair, a pathway that could be favored if the Franck Condon factors or the structure of the dimer are altered by the mutation (9 1 , 95). The state D+HA , however, is predicted to be at higher energy than P + HA because [Bphe-Bchl] is likely to be more difficult to oxidize than [BchIJz. Such a change in energetics might upset the balance between the reorganizational energy of the system and the free energy for electron transfer ( 1 1 0a), resulting in a much slower rate for the forward step (9 1 ) . Other factors, such a s alterations in the position o f orientation of the chromophores, or a decrease in the electronic overlap between them, may play a role (see below).



345

The u nusual spectroscopic and electron transfer properties of the het erodimer RCs necessitated further studies of the geometry of the pigments, to ascertain whether their orientation had been drastically altered by the mutations. Linear dichroism (LD) measurements at 1 0 K show that the shape of many of the RC bands [e.g. those at 755 nm (HA and HB), 800 nm (BA and BB), and 500 nrn (carotenoid)] are conserved between the heterodimer mutants and wild-type ( 1 6). The orientation of the Qy transi tion of the primary donor in both HisM200 � Leu and HisM200 � Phe RCs is also unchanged. Thus, no largc-scale rcorientation of the pigments takes place in either of the heterodimer RCs. Both LD and circular dichroism (CD) spectra, however, suggest that the electronic coupling between the two pigments of the primary donor is reduced in the heterodimer mutants. The loss offeatures in the CD spectra between 750 nm and 900 nm, relative to wild-type Res, evinces the reduction in coupling between all of the RC pigments. In addition to these differences, the broad absorption/LD band associ ated with the long-wavelength transition of the dimer is changed by the HisM200 � Leu and HisM200 � Phe m utations ( 1 6) . In the heterodimer RCs, it spans the region from 820 nm to about 1 000 nm and at 1 0 K displays some structure in the region around 850 nm. Another unusual feature is the presence of a negative-difference absorption change at 548 nm that appears upon D+ formation (89). The LD data indicate that the orientation of this bleaching band with respect to the C2 axis is comparable with that of the Px+ exciton band of the primary donor ( 1 6), which in wild-type RCs occurs at 600 nm ( 1 5, 1 6). The putative intradimer charge-transfer state of the heterodimer has been examined in greater detail by subpicosecond transient-difference spec troscopy (87, 95) [including photodichroism measurements (94)] and by o Stark-effect spectroscopy. Transient-difference spectra of the HisM20 ..... Leu RCs at 600 fs after excitation show a broad positive absorption centered at 650 nm that is not present in wild-type RCs (87). This band, which is attributed to a reduced bacteriochlorin, decays biphasically (with lifetimes of 1 2.0 ± 1 . 5 ps and I OO ±20 ps). The slow component is assigned to the electron transfer from HA" to QA (91 ) . The kinetics of the dominant fast decay match the kinetics for the decay of the initial excited state (called D* above) and are attributable to the reduced Bphe component of the heterodimer CT state [Bchl+Bphe-]. These assignments are supported by measurements of the time-dependent polarization of the anion band (relative to the long-wavelength absorption band of the dimer), which changes from a parallel alignment at I ps to a perpendicular one at 35 ps (87). This reasoning assumes that the transition dipoles of the pigments in the mutant do not differ drastically from those of wild-type, an assumption


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COLEMAN & YOUVAN

supported by LD measurements (discussed above) and Stark-effect measurements (below). The mechanism by which the charge-transfer states are generated within the HisM200 -+ Leu RCs has also been investigated by comparing the kin etics of the primary electron-transfer reactions at room temperature and at 77 K (95). The lifetime of the putative [Bchl + Bphe-] state of the het erodimer was observed to be temperature independent within this range, indicating that both intradimer charge recombination and forward elec tron transfer to HA are activationless. Stark-effect spectroscopy can be used to measure electrochromic effects on the excited states of the primary electron donor and the other bac teriochlorins by measuring their absorption or fluorescence spectra in the presence of a large external electric field, and hence provides a means to probe charge-transfer states (10, 13, 106). The HisM200 � Leu and HisM200 -+ Phe RCs have Stark-effect absorption spectra at 77 K that are similar to each other and to wild-type Rb. capsulatus in most regions, except for the Qy and Qx absorption bands of the special pair (52). In the region of the Qy transition of the special pair, two resolved Stark bands are observed in the HisM200 � Leu and HisM200 � Phe Res that are not present in the wild-type Stark spectrum and are not identifiable in the absorption spectrum of the mutants. Likewise, additional positive and negative Stark bands in the Qx region result from the additional Qx peak in the absorption spectrum of the mutants. The observation that the main Stark band in the Qy spectrum is red-shifted 500 em -I relative to wild type may indicate that this band reflects a CT state that is lower in energy in the heterodimer mutants (52). Stark spectra and second-derivative absorption spectra for the special pair absorption band were used to calculate l1/lapp, the magnitude of the change in the dipole moment vector from the ground to the excited state, as well as the angle () between the Qy transition moment vector (Iltrans) and b./lapp (52). The values for Rb. capsulatus wild-type (b./lapp 6 .7 ± 1.0 D and () = 38 ± 3°) are identical to those obtained for Rb. sphaeroides and Rps. viridis RCs (104, 105). Within experimental error, the values of l1/lapp and () are also approximately the same for both the HisM200 � Leu and HisM200 -+ Phe RCs (!J./lappz14 D and () 32±6°) (52). The value of !J.llapp could not be determined with greater accuracy because of low signal-to noise ratios in the second-derivative spectra of the mutants. The increase in the limiting values of b./lapp is consistent with an enhanced contribution of charge transfer to the excited state of the heterodimer Res; however, !J./lapp also might increase because of the asymmetry of the dimer itself, without an actual increase in the amount of charge transfer. Breaking the =

=



347

C2 symmetry of the special pair may cause the angle b to change as a result of the mutations, a view that is consistent with the i ncrease in dJlapp if the character of the special pair has changed from a dimer to more of a monomer (52). The cation radical state [D +], the radical pair state I[D+H"J, and the * triplet state3 [D] of the primary donor have been examined in the het erodimer mutants to probe the electronic asymmetry of the dimer and the dynamics of charge separation and recombination. Studies of the recombination of I[D+HA] to 3*[D]HA were carried out in carotenoid-free RC preparations of the heterodimer so that the triplet state of D could be measured in the absence of triplet energy transfer (36) to the carotenoids (99). Carotenoidless (crC) RCs were expressed in Rh. capsulatus by plas mid complementation of a crt - puf operon deletion background [U43b (99)]. This background was generated by introducing a pU2922 derivative containing an engineered frameshift mutation in the pufX gene into the original deletion strain U43. The cells were spotted on solid media and allowed to grow anaerobically under high-intensity illumination. Caro tenoidless mutants were isolated from the edges of the spots. These mutants were stable during purification and contain an as-yet-undefined mutation (not located on the plasmid pU2922) that renders the cells phenotypically devoid of carotenoid. EPR measurements of the cation and triplet states of the primary donor in HisM200 and HisLl73 mutants (which were generated in a crt+ background) indicate that these states in the HisM200 --- Gin and HisLI73 --- GIn chro matophores are very similar to those observed in wild-type (27). In RCs and chromatophores from the HisM200 ___ Leu and HisM200 --- Phe heterodimer mutants, the linewidth of the oxidized primary donor is sig nificantly broadened, indicating that the unpaired spin is asymmetrically distributed over the [Bchl-Bphe] dimer. Since Bchl is the easier of the two to oxidize, the cation probably resides on the Bchl half of the dimer. The yield of the photoexcited triplet state of the heterodimer RCs was found to be greatly reduced compared with wild-type, but the polarization pattern of the triplet indicates that it is still To polarized and therefore results from the recombination reaction of the radical pair after charge separation (for a discussion, see 76, 77, 1 0 1). The zero-field splitting (ZFS) parameters of the heterodimer RCs (IDI = O.02 1 O±O.OO I O cm- I ; lEI O.0060±O.OOI O cm -I ) resemble those of a Bchl a monomer in frozen solution ( 1 55), suggesting that the triplet also resides primarily on the Bchl side of the dimer (27). Kolaczkowski et al (99) used time-resolved absorbance and optically detected magnetic resonance (ODMR) to determine the basis for the =


348

COLEMAN & YOUVAN

apparently low triplet yield in the heterodimer. The radical pair state was found to have a 14 ± 1 ns lifetime in the native wild-type and carotenoidless wild-type Res, a value similar to that obtained in Rb. sphaeroides (37, 142). The carotenoid-containing wild-type RCs also show carotenoid triplet formation, due to triplet energy transfer from 3'p (99). However, the carotenoidless HisM200 � Leu RCs display a very rapid decay of the radical pair (lifetime S 2 ns) and very little residual bleaching at 860 nm (99), consistent with the reduced yield seen in the EPR experiments (27). The caro tenoid-containing heterodimer RCs monitored at 545 nm also show the same rapid decay of the radical pair, followed by a low level of carotenoid triplet formation (99). These results are tentatively explained by an increase in the singlet radical pair recombination rate, k" which is expected to ' shorten the radical pair lifetime and reduce the 3 D yield. The low triplet M200 � Leu heterodimer made it difficult to establish the yield in the His magnetic field dependence, although the value of J (representing the ex change interaction for the singlet-triplet splitting) appears to be much smaller than in wild-type RCs. Further studies of the recombination dynamics are needed to explain the properties of the triplet states in the HisM200 heterodimer mutants. Recently, a HisL173 � Leu mutation has been constructed in Rb. cap sulatus with the goal of creating an L-side (A-branch) heterodimer at the special pair (31). Like the HisM200 � Leu construction, this mutant does not grow photosynthetically, and pigment extractions of HisL173 � Leu RCs suggest that the modification causes the BchlA (PA) within the special pair to be replaced by Bphe. The HisLl73 � Leu RCs are photochemically active and have a light-minus-dark difference spectrum similar to that observed in the HisM200 � Leu RCs. The ground-state absorption spectrum is also generally similar to that of the B-branch heterodimer RCs, with the exception that the HisL173 � Leu RCs have increased absorption in the 810-860 nm region (see Figure 5). The unusual spectral properties of the heterodimer RCs have begun to shed light on the very early steps involved in charge separation in wild type RCs. The observation of spectroscopic signals reflecting a charge transfer contribution to the excited state of the heterodimer has raised new questions regarding the possible origin of such signals in wild-type RCs, where they are more difficult to discern. In the latter case, a more cryptic charge-transfer state may be present owing to the greater symmetry of the [Bchlh homodimer (87). Additional studies are needed to explore whether subtle asymmetry in the special pair dimer could be the underlying cause for the unidirectional movement of the electron along the A-branch of the RC complex.


349

REPLACEMENT OF THE AXIAL LIGANDS


TO THE MONOMERIC BACTERIOCHLOROPHYLLS

In the high-resolution Rps. viridis X-ray crystal structure, two histidine residues on the cd-helices of the L and M subunits ligate the two monomeric, or accessory, Bchls (BA and BB) (5 1 , 1 17) . The imidazole side chains of HisLI53 and HisMI80 are coordinated to the central Mg atoms of BA and BB, respectively (Figure 6). In the partially refined Rh. sphaeroides structure, however, HisLl53 does not come in direct contact with the Mg2+ atom of the Bchl ( 1 82). Researchers have not determined the functional or structural role of the monomeric Bchls with certainty. Because of thc considerable distance separating P and HA and the correspondingly weak electronic overlap between the electron donor-acceptor pair, theorists have had difficulty explaining the extremely rapid rate of electron transfer during the initial charge-separation step P*HA -+ P + HA. Consequently, the role of BA in bridging the gap between the two has become the subject of extensive experimental and theoretical investigation. Various hypotheses offer explanations of how this Bchl might participate in the primary events (summarized in 8a, 65, 110). Sequential electron-transfer mechanisms pos tulate an intermediate P + BAHA or PB,tHA ion-pair state. A single-step superexchange mechanism, in which a P+BA virtual state mixes with the excited state of P, has also been proposed. Until recently, subpicosecond measurements of the kinetics of electron transfer from p* to HA have generally supported the superexchange model because these measurements have not detected any of the BA intermediate wild-type RCs (64, 89). But Holzapfel et al (79) recently reported kinetic evidence for transient formation of B;. Presumably, the various models

.� Figure 6

This stereo pair highlights the interactions between the amphipathic cd-helices

(running along the periplasmic surface of the RC) and the monomeric Bchls, BA and BB, in

Rps. viridis. In both cases, histidines act as the axial ligands. The results of site-directed mutagenesis at these residues are discussed in the text.


3 50

COLEMAN & YOUVAN

for electron transfer can be tested by mutagenizing the protein environment around the Bchl to: (a) alter the energy levels of the P+BA or BtHA states; (b ) convert the Bchl to a Bphe [as is the case for the B-branch accessory pigment in Chloroflexus aurantiacus, in which a leucine appears to have replaced the axial histidine (12S)]; or (c) modify the electronic coupling between the Bchl and the other pigments on the A-branch. The monomeric Bchl on the B-branch (BB) does not appear to be required for electron transfer, since its removal by chemical means does not interfere with charge separation. However, BB may be involved in energy transfer to the nearby carotenoid (lOS, 143). The most prominent targets for amino acid replace ment at the monomeric Bchls a re the axial ligands to the central Mg2+ atoms: HisLl53 on the A-branch and HisM180 on the B-branch.

In Rb. capsulatus, Bylina et al (27) used directed mutagenesis and recombinant DNA techniques to change HisM180 to serine and HisL1 53 to serine and threonine and to construct a double mutation (HisLl53,M 18o � Ser). All of these mutants are photosynthetically competent, indicating that they all assemble functional RCs. Their RC pigment composition is identical to wild-type. The ground-state absorption spectra of the modified Res are generally similar to wild-type, as are the kinetics of P + QA � PQA charge recombination and the oxidized-minus-reduced (light-min us-dark) difference spectra. The only difference in the absorption spectra between the modified RCs and wild-type is in the region of the Bchl Qx transitions near 600 nm for the HisL153 mutations. In the HisLl53 � Ser Res, this band clearly splits at 77 K into two peaks separated by approximately IS nm (23). The values obtained for the P+ EPR linewidth and the triplet ZFS parameters for HisLl53 � Ser or Thr and HisM180 � Ser mutations in chromatophores are virtually indistinguishable from wild type (27). Linear dichroism and time-resolved optical measurements are needed to characterize the pigment orientation and electron transfer prop erties of these mutants. The preliminary analysis, however, shows that histidine is not required for functional assembly of the monomeric Bchls. This finding has important implications for the study of the plant Photo system II, since neither the DI or D2 RC polypeptides contains a histi dine residue at the sequence position analogous to HisLl53 or HisM180 ( 1 59). Other mutagenic replacements have been made at these two positions, but stable Res cannot yet be purified from the chromatophore membranes (27). Hence, pigment extraction data and RC spectra are not available for these mutants. The mutations HisLl53 � Leu and HisM180 � Leu are both photosynthetically competent, whereas HisM180 � Arg is not. EPR measurements on chromatophores indicate, however, that the P+ line widths and the primary donor triplet spectra are the same as wild-type. The ground-state absorption spectra of the Res in these more labile



351

mutants are being examined in chromatophores (without interference from the light-harvesting complexes) by using a recently developed background strain of Rb. capsulatus (28) that lacks both LH I and LH II (see below). This type of in situ optical spectroscopy has allowed researchers to see the RC absorption bands in the near-infrared without overlapping absorption from the light-harvesting pigments and provides a good example of how genetics can be exploited to assist in spectroscopic characterization. Fur ther characterization of the HisLI53 --t Leu mutations is particularly impor tant, since the RCs are evidently capable of catalyzing a photochemical charge separation (27). If this mutant possesses a Bchl at the monomer position on the A-branch, then the structural criteria responsible for bac teriochlorin pigment binding in the RC must be reexamined. If a Bphe is inserted at this position, then the mutant may provide a crucial test for models of the initial charge separation, since the energy of the state P + Bphe- relative to p* may be much lower than the state P+Bchl-. THE SPECTROSCOPIC RED SHIFT OF THE PHOTOACTIVE BACTERIOPHEOPHYTIN

The two Bphes in the Rps. viridis and Rb. sphaeroides RCs are chemically identical within each species and are located in approximately symmetrical positions with respect to the C2 axis (51, 117, 182). Because their spectral properties are not correspondingly identical, however, the observed differ ences must be the result of local environmental interactions. At low tem perature, the two Bphes (HA and HR) can be distinguished optically by the orientation dependence and wavelength position of their Qx and Qy transitions (14, 35, 94, 158, 165). The Bphe absorbing at longer-wave lengths is the one that is reduced as a result of the photochemical charge separation (59,78, 84, 92, 93, 141, 16 7) . Analysis of the absorption spectra of single crystals from Rps. viridis (containing only the menaquinone QA) (97, 191) and modeling of the optical transitions based on the three dimensional structure of the RC (98) indicate that the long-wavelength, photoactive Bphe is nearest to QA. This Bphe is therefore the one desig nated HA• In Rb. capsulatus, the Qx absorption maximum of HR is at 531 nm at 77 K, whereas the Qx band of HA is centered at 546 nm (26). One of the most striking structural asymmetries in the X-ray crystal structures of Rps. viridis and Rb. sphaeroides RCs is the presence of a glutamic acid residue (GluL104 on the B-helix) within hydrogen-bonding distance to the ring V C9 keto group of HA (Figure 7) (51, 117, 182). This residue is conserved in a number of LfDl-subunit sequences from various organisms, including Rb. capsulatus (sec 26, 116 for discussion). ENDOR (electron nuclear double resonance) (62), resonance Raman (9), and FT-


3 52

COLEMAN & YOUVAN

Figure 7 The interactions of two critical amino acid residues in Rps. viridis with the photo active Bphc (HA) and the primary quinone (QA) are displayed. Mutagenesis and spectroscopy have been used to investigate the role of glutamic acid residue L l04 in deter mining the directionality of electron transfer and in shifting the Qx absorption band of HA in Rb. capsulatus. Mutagenesis has also been used to assess the role of tryptophan M250 in stabilizing quinone binding and in facilitating electron transfer between HA and QA'

IR (Fourier transform-infrared) ( 1 2 1 ) measurements have indicated that

GiuLlo4 interacts with the C=O group of HA• Such an interaction could contribute to the red shift of the optical absorption bands of this pigment. Because no symmetry-related ionizable residue equivalent to GiuLl04 exists on the B-branch of the bacterial RC, questions were raised as to whether GiuLl o4 might be important for directing the photoelecron down the A branch ( 1 1 7). Thc "branching ratio" of the primary electron transfer rates along the A-branch versus along the B-branch favors the former by about an order of magnitude ( 1 20), but the search continues for an explanation of this apparent unidirectionality ( 1 20, 1 37). To test whether GiuLl04 is responsible for the spectroscopic red shift of HA, three mutations were introduced at this position: one to test the effects of substituting a side chain that is a weaker proton donor (GluLl04 � Gin), another that is incapable of forming a hydrogen bond (GluLl04 � Leu), and a third that is positively charged at the physiological pH (GluLl04 � Lys) (26). The GiuLI04 � Lys mutant does not grow photosynthetically and does not appear to assemble RCs. However, both the GluL104 � Gin and GiuLl04 � Leu mutants are photosynthetically competent and do assemble functional RCs. A comparison of the 77-K absorption spectra of the purified RCs reveals a progressive blue shift in the Qx absorption band of HA: from 546 nm in wild-type to 540 nm in the GJuLl04 -> Gin RCs, to a single peak (overlapping with HB) at 534 nm in the GiuLl04 � Leu RCs. The Qx transitions are not degenerate in the GiuL l o4 � Leu RCs; however, the lO-K absorption spectrum still shows a splitting of several



353

nanometers between the two peaks (Figure 8) ( 1 6). The greater width of the HB peak relative to the HA peak also remains in the mutants ( 1 6). Room-temperature measurements of the electron-transfer kinetics indicate that the mutations have only a small effect on thc rate of reduction of either HA or QA (26). The initial electron transfer step (P*HA � P + HAJ is less than two fold slower in the mutants, and the second step (P+ HA:QA � P+HAQA:) is only marginally slower. The overall charge sep aration has a quantum yield of essentially unity, and no evidence suggests electron transfer to HB• The 1 2-ps Qx bleaching spectrum of 8phe in the

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(10 K) ground-state absorption spectra of purified RCs altered a t

GluLlo4. In wild-type RCs, asymmetries in the protein structure result in absorbances at

slightly different wavelengths for HA and HB• Wild-type Rb. capsulatus (short-dashed line) HA absorbs at 545.5 nm and HR absorbs at 529.5 nm (Qx transitions). There is a 3-nm blue shift in HA absorption when GiuLlo4 is mutated to glutamine (dotted line) and a 9-nm blue shift whcn it is mutatcd to leucine (solid line). This latter value corresponds to about two thirds of the difference in absorption (529.5 nm and 545.5 nm) between HB and HA in wild type. A spectrum of wild-type Rhodobacter sphaeroides (long-dashed-dotted line) is included to illustrate differences in HR absorption that can be assigned to interspecies sequence differences. Reprinted with permission from Breton et al ( 1 6).


354

COLEMAN & YOUVAN

GlULl04 � Leu RCs also shows a blue shift (relative to wild-type) that is analogous to the one observed in the 77-K absorption spectrum, thus confirming that HA is the photoactive electron acceptor whose Qx band is red-shifted by interacting with GluLl04 . Linear dichroism measurements of RCs from GluL104 � Gin and GiuLl04 � Leu in the region of 520 to 560 nm do not indicate any changes in the orientation of the Qx transition of HA relative to the C 2 axis of the RC as a result of the mutations {J 6). The measured values for the EPR linewidth of P+ and the ZFS parameters of the primary donor triplet state are the same as in wild-type (27). The conclusion that can be drawn from these studies is that the putative hydrogen bond between GiuLlo4 and HA contributes substantially (but not completely) to the red shift in the HA absorption but that GiuLlo4 does not significantly influence either the primary photochemistry or the direction ality of electron transfer within the RC. The basis for the asymmetry in electron conduction must therefore lie in some other facet of the RC structure. THE QUINONE ELECTRON ACCEPTORS

The primary and secondary quinones (QA and QB, respectively) occupy binding sites composed primarily of residues from the ends of the D- and E-helices, as well as from the de interhelical regions. The two quinones are related to each other by the C 2 symmetry axis of the RC and are bridged by the nonheme Fe (3, 33, 5 1 , 1 1 7). In Rps. viridis, QA is a menaquinone, M K9 (the subscript denotes the number of isoprenoid units in the tail) and QB is a ubiquinone, UQ9 (66, 1 1 7). In Rh. capsulatus and Rh. sphaeroides, both quinones are UQlO (3, 42, 63, 1 1 1 ). Although QA and Q8 are similar or identical in their chemical structure, their functional properties are profoundly influenced by the protein matrix. Thus, QA acts as a tightly bound one-electron acceptor that does not appear to be directly protonated following photochemical reduction, whereas QB is a mobile two-electron, two-proton redox component (see 6 1 , 63, 125, 1 78, 1 80 for discussion). Photosynthetic herbicides inhibit electron transfer in the RC by competing with QIl for the secondary binding site (53, 160, 178). Electron Transfer to the Primary Quinone: QA Binding and Reduction

The X-ray crystal structure of the Rps. viridis RC shows that the keto carbonyl oxygen atoms of QA are positioned to make hydrogen bonds with the peptide NH group of AlaM258 on one end and with the N81 nitrogen atom of HisM217 on the other end (51, 1 1 7). HisM217, one of five protein



355

ligands to the Fe atom, forms a bridge between QA and the metal center. The crystal structures also show that a conserved tryptophan residue (M250 in Rps. viridis and Rb. capsulatus, M252 in Rb. sphaeroides) is positioned within van der Waals contact distance of both HA and QA (also displayed in Figure 7). In the high-resolution Rps. viridis structure, the M K9 ring is nearly coplanar with the TrpM250 indole side chain, separated from it by a distance of 3.1 A (51). The location of TrpM2S0 with respect to HA and QA has prompted speculation that it might be involved in promoting both tight binding ofthe quinone [owing to a favorable stacking interaction between the rings ( 1 1 7)] and fast electron transfer from HA" to QA [by enhancing the electronic overlap between the Bphe and quinone (136)]. The latter hypothesis (superexchange) has been proposed to explain the rapid rate of electron transfer [kQ = (200 pS)- 1 at 295 K (89-91, 93)] over the � 14 A center-to-center distance and � lo A edge-to-edge distance between HA and QA (136). According to the superexchange hypothesis, the indole group of TrpM 250 might act as a bridge between the orbitals of the electron donor (HA) and the electron acceptor (QA) by means of quantum mechanical mixing with the donor and acceptor states (providing the virtual states HtTrp- or Trp+ QA)' A total of seven directed mutations (Phe, Leu, Met, Thr, Val, Glu, and Arg) have been introduced at TrpM2S0 to determine their effects on QA binding and reduction (40). Only one of these mutants (Phe) is capable of growing photosynthetically. Isolated RCs from the TrpM250 --> Phe mutant retain a large fraction of QA after the purification procedure, whereas RCs from the other TrpM250 mutants do not appear to contain QA, since the long-wavelength absorption band of P does not bleach under continuous actinic illumination. However, all of the genetically modified Res are capable of performing charge separation when a sufficiently high con centration of quinone is added to the buffer solution. Previous work with Rb. sphaeroides has shown that one can extract ncarly all of the endogenous quinone from wild-type RCs with a combination of detergent [LDAO (N,N'-Dimethyldodecylamine-N-oxide)] and o-phenanthroline (126, 175). Photochemical reduction of quinone can be restored by reconstituting the quinone-free preparations with either the native quinone or suitable quinone derivatives (69-73, 126, 175). Quinone reconstitution experiments in Rh. capsulatus, using quinone-extracted wild-type and TrpM2S0 --> Phe RCs as well as the other quinone-depleted mutant RCs, indicate that the apparent affinity of the RC for quinone (expressed as an apparent dissociation constant, K'o) depends strongly on the nature of the side chain replacement at M 250 (40). Wild-type Rh. capsulatus RCs have a K'o for 2-methyl- I ,4-naphthoquinone (MKo) of < 1 flM, which is consistent with that reported for wild-type Rb. sphaeroides RCs (69). The apparent

356

COLEMAN & YOUVAN

affinity of the TrpM 2 50 -+ Phe Res for MKo (2 flM) is only slightly lower than in wild-type, consistent with a conservative aromatic replacement (40). The other nonaromatic replacements have relatively lower affinities for MKo (K'D 8-1 3 flM for TrpM250 -+ Met, Thr, or Leu; 200-270 flM for Glu, Val, or Arg). Thus, one possible explanation for the inability of most of the mutants to grow photosynthetically is that the QA site is not fully occupied in vivo. To determine whether mutagenesis of TrpM250 affects electron transfer as well as quinone binding, the electron-transfer rate constant kQ was measured by observing the kinetics of the recovery of the Qx absorption band of HA at 545 nm (39). The 545 nm band is bleached when a brief excitation flash generates the state P + HA (e.g. 26). Wild-type Res in which the native UQIO has been extracted and replaced with M Ko have a value for kQ of ( 1 70 ps) - ' at 85 K (39). This value is similar to that obtained for o the native wild-type Res ( 1 35 ps)- ' . In contrast, the TrpM2s -+ Phe Res reconstituted with UQ show a reduction in kQ by a factor of about 7 [(880 ps)- ' ] . The aliphatic substitution TrpM250 -+ Leu slows the rate by a factor of about 1 6 [(2700 ps)- ' ] . The inhibitory effects on kQ are approximately the same at 280 K. The rates for HA -+ QA electron transfer in both wild type Res and the mutants appear to be activationless, since they exhibit a weak increase when the temperature is lowered (8, 90). Although the decrease in kQ as a result of the TrpM250 -+ Phe and TrpM250 -+ Leu mutations is consistent with the possibility that TrpM250 acts as a super exchange mediator ( 1 36), other explanations cannot be ruled out because the decrease in kQ might arise from changes in the direct coupling between HA and QA (owing to an increase in the separation between the two molecules or a shift in their relative orientations), as w(}ll as from changes in the superexchange coupling (owing to the nature of the intervening amino acid residue). Additional information from EPR, ENDOR, M ARY (Magnetic Field Effect on Reaction Yield), and RYDMR (Reaction Yield Detected Magnetic Resonance) measurements are needed to address this question.


=

Mutations Affecting QB and Herbicide Binding

The secondary quinone (QB) binding site is composed primarily of amino acid residues from the L-subunit (Figure 9) (3, 51). Electron transfer from QA to QB occurs in approximately 1 00-200 flS ( 1 66, 1 77), and the electron transfer rate is not greatly affected by extraction of the Fe2 + atom or its replacement with other divalent metals (49). Genetic studies of the secon dary QB binding site have focused on the molecular mechanism of heribicide resistance because this subject is relevant to PS II electron transfer ( 1 52, 1 6 1 ). Many herbicides, such as atrazinc (2-chloro-4-ethyl-



Figure 9

357

secondary quinone (QB) is held in i ts binding site by a number of interactions imidazole group of HisLl9o, which also serves as a bridge to the Fe2+ atom. The other carbonyl oxygen is hydrogen bonded to the hydroxyl side chain of ScrL223• In this high-resolution Rps. viridis structure, a second hydrogen bond with the N H group of GlyL225 is also possible. Phenylalanine (PheL 2 1 6) and isoleucine (I/eL 22 9) side chains lie in opposite sides of the Qs ring plane. Herbicides also bind at this site, and many mutations conferring herbicide resistance occur at these structurally important residues (see text) . The

with the L-subunit. One of the QB carbonyl oxygens is hydrogen-bonded to the

amino-6-isopropyl-amino-s-triazine), act as competitive inhibitors of QB in both bacterial RCs and PS II by displacing the quinone from its binding site (e.g. 20, 150, 152). Mutations conveying herbicide resistance enable the RC to increase its relative affinity for quinone over the herbicide (20, 1 3 0 1 50, 1 52). The QB domain of the RC protein serves as an interesting model system for membrane protein engineering and biotechnology applications, since it offers the opportunity to modify quinone and inhibitor binding in a prokaryotic system by relatively simple�gel!etic techniques. The first directed mutations introduced into the purple bacterial RC were aimed at identifying the role of HeL229 , a position at which a spontaneous IleL229 -+ Met mutation has been found to convey triazine herbicide resistance in Rb. :,phueroides (67, 130). In Rb. cupsulatus, oligonucleotide-mediated mutagenesis was used to change this amino acid (which comes in contact with QB in the Rps. viridis and Rb. sphaeroides structures) to all of the other amino acids except proline and phenylalanine (29). Reaction centers are assembled in all of these mutants; however, 1 0 of the 1 7 mutants (Gly, His, Tyr, Trp, Asn, Asp, Gin, Glu, Arg, and Lys) do not grow photosynthetically. The other seven mutants have varying levels of pho tosynthetic growth [Ile (wild-type), Val, Ala, Leu, Met > Thr, Cys, Ser]. Six of the seven photosynthetically active mutants show varying degrees ,


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of atrazine resistance (Met, Leu > Ser, Ala, Thr, Cys > Val, Ile), but none shows increased sensitivity to atrazine. (However, this is not true for some other herbicides; see below.) In general, hydrophobic residues of moderate size function best at Ilel229 . The susceptibility of a given mutant to a particular herbicide depends on the mutual interaction between the amino acid side chain(s) and the functional groups on the inhibitor. Differential resistance to certain herbi cides has been observed in plants and bacteria ( 1 30, 1 49, 1 6 1) and, in most cases, probably reflects the fact that inhibitor binding sites for different classes of herbicides overlap but are not necessarily identical (5 1 , 1 1 7, 1 30, 1 50, 1 62). For inhibitors with very similar chemical structures, differential resistance may arise from a specific, small-scale interaction between a substituent group on the herbicide molecule and a nearby amino acid side chain. Photosynthetic growth assays, for example, indicate that the valine mutant at L229 is much more sensitive to s-triazine herbicides, such as atrazine or terbutryne (2-tert-butylamino-4-ethylamino-6-methyIthio-s triazine), than are the leucine or methionine mutants (25). However, RCs containing the valine or isoleucine side chains are less sensitive to ametryne (2-ethylamino-4-isopropylamino-6-methylthio-s-triazine), prometryne [2,4bis(isopropylamino)-6-methylthio-s-triazine], and terbutryne than are mutants possessing smaller amino acid side chains such as alanine, serine, or cysteine. Likewise, the leucine mutant (compared with wild-type) is resistant to ametryne and terbutryne, but sensitive to prometon [2methoxy-4,6-bis(isopropylamino)-s-triazine] and prometryne. The differ ential effect in this mutant may result from the presence of an ethyl amino group on ametryne and terbutryne, which is replaced by an isopropylamino group on prometon and prometryne. Site-directed mutations at PheL216 (Thr, Leu, Val, and Pro), a residue also in close proximity to the QB ring, generate differential responses to various triazine herbicides as well. A spontaneous mutation at this position in Rps. viridis (Phel216 � Ser) pro duces an altered Qs Fe2 + EPR signal in chromatophores ( 1 50). EPR analy sis of the Ilel229 � Met mutation in Rh. sphaeroides RCs suggests that this mutation shifts QB farther away from HisLl 90 ( 1 30), whose imidazole group connects QB with the nonheme Fe. Displacements of Qs and/or herbicide within the pocket as a result of steric hindrance or loss of hydrogen bonds could explain these varied effects among different mutation/inhibitor com binations. The somewhat atypical response of the Rh. capsulatus mutants to atrazinc, compared with the other s-triazine herbicides, suggests that chemical alteration of the chIoro substituent might yield new herbicides with new specific ities (25). Since the three-dimensional structure of the PS II RC is not yet known, some of the mutations that confer herbicide resistance in plants have been



359

investigated by characterizing "analogous" mutants in bacteria. The x ray crystal structure of the Rps. viridis RC indicates, for example, that the side chain of SerL223 can form a hydrogen bond with either a QB carbonyl oxygen atom (5 1 ) or with the aminoethyl group of the herbicide terbutryne (5 1 , 1 1 7). Because of the suggestion that SerL223 might be analogous to Ser-264 in the D l subunit of PS II ( 1 1 6), a site at which a Ser264 --t Ala mutation confers herbicide resistance (58), two site-directed mutations have been introduced at SerL223 in Rb. capsulatus to study their effects on the bacterial RC (25). The SerL223 --t Ala mutant in Rb. capsulatus, how ever, cannot grow photosynthetically, unlike the Ser264 --t Ala mutation in plants. The response to herbicides in the SerL223 --t Asn mutant is very similar to that in wild-type (i.e. normal photosynthetic growth, but herbicide susceptible). Thus, a hydrogen-bonding side chain at L223 ap pears essential for QB function in Rb. capsulatus. An alternative side chain or backbone hydrogen bond might be available for this purpose in plants. In Rps. viridis, the spontaneous mutation SerL223 ---t Ala, which confers herbicide resistance, is accompanied by an ArgL217 --t His mutation ( I SO). Spontaneous mutations that generate herbicide resistance in Rb. cap sulatus were also identified at several other sites in the QB pocket, many of which were previously unrecognized. The nucleotide changes were deter mined by extracting and sequencing the pU2922 plasmids from the resist ant mutants, and thc phenotype was verified by returning the plasmids to the U43 deletion background. These mutations include GlyL228 ---t Val and Arg, and ThrL226 -+ Ala and Met (25). Spontaneous mutants at TyrL222 , which is outside the immediate environment of QB, have been isolated from Rb. sphaeroides ( 1 30) and Rps. viridis ( 1 50). A TyrL222 ---t Phe mutation in Rps. viridis may indirectly confer resistance by eliminating a potential hydrogen bond with AspM43 , thereby rearranging the structure of the protein ( 1 50). MUTATIONS INVOLVING OTHER COMPONENTS OF THE PHOTOSYNTHETIC APPARATUS .

Electron Donation from Cytochrome

C2

to the Oxidized

Special Pair

TyrosineLl62 occupies a position in the X-ray crystal structure of Rps. viridis approximately halfway between the special pair Bchls and the nearest heme of the bound cytochrome subunit (5 1 , 80, 1 1 7). This tyrosine is conserved in all of the purple bacteria for which DNA sequence infor mation is available and occurs at an analogous position in the X-ray crystal


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structure of the Rb. sphaeroides RC (1 82), which has a mobile rather than a bound cytochrome. Since the phenolic side chain of this residue might be involved in facilitating electron transfer from the reduced cytochrome C2 to the oxidized special pair (51, 80), TyrLI62 was changed to Lys, Glu, Val, and Ser by site-directed mutagenesis in Rb. capsulatus (83). Plate assays indicate that only the TyrLl62 ---+ Lys mutant does not grow photo synthetically. Since Rh. capsulatus appears to be able to grow in the light without cytochrome C2 (46, 138), however, measurements of the yield of cytochrome oxidation in vitro were necessary. Assays of horse-heart cytochrome c oxidation following a series of subsaturating flashes do not indicate any significant difference between the mutants and wild-type (83). Furthermore, EPR measurements of the P+ linewidth and the triplet ZFS parameters are very similar among all four mutants and wild-type. The only significant difference is the observation of a 25-mV increase in the P/P+ midpoint redox potential of the special pair in RCs from the TYrLl62 ---+ Lys mutant. Nevertheless, the lethal effect of the TyrLl62 ---+ Lys mutation is most likely due to disruptive structural changes on the peri plasmic side of the membrane, resulting in an altered orientation of the RC or a modification of its interactions with other protein subunits (e.g. the cytochrome C2, the cytochrome bCI oxidoreductase or LH I). Time resolved optical measurements are needed to characterize the effects of these mutations on the electron-transfer kinetics. Bacteriochlorophyll Binding in the Light-Harvesting I Antenna

The B880 core antenna and B800-850 peripheral light-harvesting antenna complexes of many of the Rhodospirillaceae (referred to here for Rb. capsulatus as the LH I and LH II complexes, respectively), are composed of pigment-binding proteins whose function is to collect and transfer excitation energy to the RC with high efficiency (for reviews, sec 1 8, 55, 1 92). The LH I complex may also have a role in orienting the RC complex in Rb. capsu!atus (81). The B800-850 complexes from various organisms have been crystallized (4, 38, 1 09, 1 69), and the three-dimensional structure is currently being determined. The identity of the amino acid residues that might be responsible for binding Bchl has been inferred from comparisons of conserved sequences from a number of species (e.g. 1 92). Theiler & Zuber ( 1 54) have proposed that the IX and fJ polypeptides of the LH I complex each bind one Bchl molecule at a histidine residue within the conserved sequence Ala-X-X-X-His. The histidine may provide the axial ligand for the Bchl (21 , 140, 1 53), and the alanine side chain at the N-4 position (where N is the position of the histidine) may be in van der Waals contact with the Bchl ring ( 1 54). Directed mutagenesis of both Hisa32 ---+



361

Asn, Asp, GIn, Thr, Arg, and Pro, a s well a s Alaa28 � Gly, Ser, Cys, Val, Asp, Glu, Phe, and His on the IX structural polypeptide of Rh. capsulatus was performed to determine the role of these two residues in binding Bchl (28). All of the Hisa32 mutants grow poorly under photosynthetic conditions, and the absorption spectra of chromatophores indicate that these mutations, carried on the pU2922 plasmid, result in the loss of the LH I complex from the membrane in either the U43 deletion background (RC LH 1- LH 11-) or the U 1 5g deletion background (RC- LH 1- LH 11+) (28). All of the mutants assemble functional RCs, however, and because the RC genes are known to bc transcribed downstream of the LH I genes, the disappearance of the LH I complex cannot result from a transcriptional blockage. Moreover, since the phenotype of the mutants is unchanged even in an LH 11+ background, the lack ofLH II cannot be solely responsible for the loss of LH I. The disappearance of the LH I Bchl absorption peak in all of the Hisa 3 2 mutants suggests that the structural requirements for Bchl ligation in the LH I IX site are much more strict than in the reaction center Bchl binding sites (see above). Mutations at Alaa28 , however, can be fully tolerated in most cases in which the molar volume of the substituting side chain is smaller than that of valine (28). The mutants Alaa28 --+ Ser, Cys, and Gly grow normally under photosynthetic conditions and have wild-type levels o f LH I in either the LH II+ or LH n- background. Alaa 2 8 � Val mutants have reduced levels of photosynthetic growth and expression of LH I, which suggests that unfavorable steric factors destabilize the protein in this mutant. Molar volume cannot be the only limitation on the nature of the N-4 residue, however, since an Alaa28 --+ Asp mutation results in a loss of LH I, similar to the Hisa32 mutants discussed above. Ionization of this Asp may interfere with stable insertion of the complex into the membrane bilayer. The mutations Ala"28 --+ Phe, His, and Glu also result in the loss of LH I and reduced levels of photosynthetic growth. The tentative conclusion in the absence of direct structural information is that steric hindrance to Bchl binding limits the choice of amino acid at the N-4 position to small residues such as Ala, Ser, Cys, and Gly. The fact that photochemically active RCs can be assembled into the membranes of mutants lacking both of the LH complexes (such as Alaa28 --+ Glu in the U43 background) provides an interesting opportunity to gen erate a novel genetic background, i.e. one that is completely free of absorp tion from LH I and LH II (28). Using this new genetic construction, one can obtain spectra of the RC in situ (Figure 1 0). By cloning previously constructed RC mutants (containing mutations in the L- and/or M-subunit genes) into such a background, one can observe genetically modified RCs

362

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2 o

li:


a:

o en CD « w >

5w

a: 200 300 400 500 600 700 800 900 WAVELENGTH (nm) Figure 10 In situ optical spectroscopy of membrane-bound RCs in strains of Rh. capsu/atus that have been engineered to block LH expression. For comparison, spectrum A shows the room-temperature ground-state absorption spectrum of purified wild-type RCs. The protein and all six tetrapyrrole pigments contribute to absorption at 280 nm and 380 nm, respectively, while the near-infrared bands primarily result from specific prosthetic groups: 850 nm (PA + PB), 800 nm (BA + BB)' and 760 nm (HA + HB). These near-infrared absorbances can be observed (spectrum B) in the spectrum of chromatophore membranes [e.g. strain U43(Ala"28 --+ Glu)], when LH expression is inactivated by point mutations.

directly in chromatophores or whole cells even when the RCs are too labile to be extracted from the membrane. CONCLUDING REMARKS

Characterization of point mutations in the photosynthetic apparatus of the purple bacteria has contributed new information regarding the struc tures and processes involved in capturing light energy and converting it into a transmembrane charge separation. However, the fundamental reasons for the rapid rate of electron transfer within the RC, the high quantum yield of the forward reactions, and the unidirectionality of elec tron transfer are still unknown. The role of the protein matrix in facilitating and directing electron transfer, either by direct interactions with the chro-



363

mophores or by subtle perturbations in the overall symmetry of the RC structure, undoubtedly requires a more global approach to protein en gineering, if it is to be comprehended in its entirety. Future studies of protein structure and function in the bacterial RC will likely explore even larger questions, such as: (a) the effects of large-scale amino acid asymmetry on the electronic structure of the chromophores and on the electron-transfer dynamics; (b ) the role of entire helices in chromophore binding; and (c ) the possibility of redirecting electron trans fer onto the normally inactive B-branch. Systems for generating and screening large numbers of bacterial mutants (containing highly complex libraries of mutations) are already being developed. The photosynthetic apparatus offers the advantage of having built-in "reporter groups" (i.e. intensely colored chromophores), which, as a number of point m utations have demonstrated, are capable of signalling geneticially introduced modi fications in the pigment-protein structure. Application of new techniques such as digital imaging spectroscopy, whereby spatial and spectral infor mation can be gathered directly from bacterial colonies ( 1 8 1), will make feasible these more ambitious routes to understanding the light reactions of photosynthesis. ACKNOWLEDGMENTS

This work was supported by the National Science Foundation (DMB8609614), the United States Department of Agriculture (87-CRCR1 -2328), the National Institute of Health (R I GM42645A), and a NSF Postdoctoral Fellowship to W. J. Coleman. D. C. Youvan gratefully acknowledges receipt of an Atlantic Richfield Professorship and a Cabot Solar Energy Award. We thank Johann Deisenhofer, Dewey Holten, and Edward J. Bylina for helpful comments and a critical reading of the manuscript. Literature Cited

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High-resolution structures of photosynthetic reaction centers.

Photosynthetic electron transport in genetically altered photosystem II reaction centers of chloroplasts.

Structures of bacterial photosynthetic reaction centers.

ITO hybrid nanostructure.

Rapid-flow resonance Raman spectroscopy of bacterial photosynthetic reaction centers.

Primary photochemistry of reaction centers from the photosynthetic purple bacteria.

Reengineering the optical absorption cross-section of photosynthetic reaction centers.

Femtosecond spontaneous-emission studies of reaction centers from photosynthetic bacteria.

Genetically modified photosynthetic antenna complexes with blueshifted absorbance bands.

Electron donors and acceptors in photosynthetic reaction centers.

Proton transfer in reaction centers from photosynthetic bacteria.

Localization of photosynthetic reaction centers by antibody binding to chromatophore membranes from Rhodopseudomonas spheroides strain R26.

Computer simulations of electron-transfer reactions in solution and in photosynthetic reaction centers.

Mechanisms of long-distance electron transfer in proteins: lessons from photosynthetic reaction centers.

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Genetically modified.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells.

Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers.

A single residue controls electron transfer gating in photosynthetic reaction centers.

Functional type 2 photosynthetic reaction centers found in the rare bacterial phylum Gemmatimonadetes.

A meta-analysis of the impacts of genetically modified crops.

Metabolomics of genetically modified crops.

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Spatiotemporal patterns of non-genetically modified crops in the era of expansion of genetically modified food.