Proton transfer in reaction centers from photosynthetic bacteria.

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Annu. Rev. Biochem. 1992. 61 :861�96 Copyright © 1992 by Annual Reviews Inc. All rights reserved

PROTON TRANSFER IN REACTION

Annu. Rev. Biochem. 1992.61:861-896. Downloaded from www.annualreviews.org by Florida State University on 05/13/13. For personal use only.

CENTERS FROM PHOTOSYNTHETIC BACTERIA

M. Y. Okamura and G. Feher Department of Physics, University of California-San Diego, La Jolla, California 92093-0319 KEY WORDS:

quinone, electron transfer, site-directed mutagenesis, protonation, photosynthesis

CONTENTS INTRODUCTION AND PERSPECTIVE . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . STRUCTURE OF BACTERIAL REACTION CENTER . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . QUINONE CHEMISTRY . . . . . .. . .... .. . ... . .... . . . ... . . . . . . . . . . . . ... ... . . . . . .. . . ... . . . . .. . . . . . . .. . .

QUINONE REDUCTION CyCLE................................................................ EXPERIMENTAL APPROACHES TO THE PROBLEM OF PROTON TRANSFER . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . .. . . .. .... . . . . . . . . . Spectroscopy .. . . . . . . . . . . . . . . . . . . ... . ... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . , . . . .. . . . . . . . . . Stoichiometry of Proton Uptake. . . . . . . . . . .. . . . . . . . . ....... ... . . . . . . . . . . . . . . ... . . .. . . . . . .. . . . . . . Rates of Electron Transfer and Proton Uptake . .. . . ... . ... . . . . . .. . . . . ............... . . ..... Effects of Site-Directed Mutagenesis on Electron and Proton Transfer Rates . . . . . . . . Electron and Proton Transfer Equilibria. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . MECHANISM OF PROTON TRANSFER................................................. ...... Sequence of Electron and Proton Transfer Steps . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . Structural Models for the Proton Transfer Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dynamic Models of Proton Transfer.. ... . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Energetics of Proton Transport . . .. . . . . . ... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUMMARy ........................................................... .......... ............. ..........

861

863

865

868

869 870 871 873 875 880 884 885 885 886 888 891 893

INTRODUCTION AND PERSPECTIVE In purple photosynthetic bacteria light energy is transformed into chemical energy by the action of a light-driven proton pump coupled to electron

0066-4154/92/0701-0861$02.00

861


862

OKAMURA & FEHER

transfer. A central role in this process is played by the bacterial reaction center (RC), a membrane protein that absorbs photons, performs the initial rapid electron transfer reactions and is the site for the initial proton uptake reactions of the proton pump (reviewed in references 1-4). The key reactions that occur in the RC involve the two-electron reduction and concomitant binding of two protons from the cytoplasm side of the membrane by a bound quinone, QB , on the RC (Figure 1). The doubly reduced quinone subsequently dissociates from the RC and is reoxidized by the cytochrome ble\ complex, releasing protons on the periplasmic side of the membrane. The net result of these reactions is the vectorial transport of protons across the membrane, driven by electron transfer, as proposed by Mitchell (5). This proton transport produces a pH gradient that drives ATP synthesis (reviewed in reference 6). The transmembrane proton-pumping function of the RC has been known since the initial observation by Baltscheffsky & von Stedingk (8) of proton uptake by chromatophores from Rhodospirillum rubrum. Further studies of this proton uptake in intact membranes (9-1 1) established that it was associ ated with electron transfer to the secondary acceptor species (now known to be QB)' An important advance was the isolation of RCs by Reed & Clayton (12) and subsequent further purification (13) and characterization of its electron transfer components (reviewed in 14). Proton uptake measurements on isolated RCs led Wraight to propose the basic ideas for proton transport in RCs, i.e. proton uptake by the doubly reduced quinone and the involvement of acidic residues from the protein in this process (15). However, the mo1ecu-

peri plasm

Figure 1

Schematic representation of electron and proton transfer in bacterial photosynthesis.

Electron transfer steps arc indicated by solid lines, proton transfer steps by dashed lines, and

diffusion steps by dotted lines. Proton transfer is coupled to electron transfer via the protonation of

QB2-

in the Re. Reproduced from reference

7 with permission.

PROTON TRANSFER IN REACTION CENTERS

863

lar details of these mechanisms have only recently begun to be elucidated. Two major developments have contributed to our present understanding:

(a) Rhodop seudomonas viridis by Deisenhofer et al (16) and from Rhodobacter sphaeroides by Allen et al (17) and Chang et al (18), and (b) site-directed mutagenesis on RCs from Rb. sphaeroides by Paddock et al (7, 19) and Takahashi & Wright (20), which was guided by these structures. The X-ray the determination of the X-ray crystal structure of the Res from


crystal structure revealed that QB is located in the interior of the protein, out of contact with the aqueous solution, and suggested the possibility that protonatable amino acid side chains from the protein were responsible for proton transport to QB. Site-directed mutagenesis of several of these residues to nonprotonatable groups resulted in loss of proton transport to QB and conclusively demonstrated that the protein plays an important role in proton transport. These findings have led to renewed interest in the mechanistic and

structural interpretation of earlier measurements of light-induced proton up

take and the pH dependence of electron transfer rates and equilibria, and they have encouraged new measurements that are sensitive to proton transfer, e.g. light-induced electrogenicity and infrared (IR) spectroscopy. In this review we discuss the results of biophysical measurements made on native and mutant RCs, which address the following questions:

(a) What proton transfer (b) Which amino acid residues are involved in these steps? (c) What are the rates and energetics for these steps? (d) What are the

steps are involved?

mechanisms of proton transfer? These questions are similar to those posed for other proton transport proteins in biological membranes, e.g. bacteriorhodop sin

(21), FIFo ATPase (22), lac permease (23), and cytochrome oxidase (24). (1, 25, 26), electron transfer reactions (27), and quinone chemistry (28), as well as on proton transfer processes in biology (29, 30). The electron and proton transfer in bacterial RCs has been recently discussed (31, 32). Recent reviews have been published on the RC structure

STRUCTURE OF BACTERIAL REACTION CENTER The RC from

Rb. sphaeroides (shown in Figure 2) contains three protein (33, 34). The Land M subunits each have five

subunits, L, M, and H

membrane-spanning helices, which form the core of the Re. The H subunit contains only one membrane-spanning helix. Most of the H subunit consists of a globular protein localized on the cytoplasmic side of the membrane covering the quinone Fe complex. The H subunit is asymmetrically located in Res from both Rps.

viridis (16) and Rh. sphaeroides (33), forming a cap over

the QA region of the RC. This cap may serve to isolate QA from the exterior aqueous environment.


864

OKAMURA & FEHER

Figure 2

Structure of the RC from Rh.

sphaeroides. The cofactors (dark lines) are embedded in (L, M, H) containing 11 membrane-spanning a-helices and a globular part on the cytoplasmic side. Electron transfer proceeds from the primary donor 0 across the membrane along the A branch via QA to QB' Proton transfer proceeds from the external aqueous phase to QB' which is buried in the protein. The doubly reduced QBH2leaves the pocket presumably by a pathway along the isoprenoid tail. Modified from reference 33 with permission. the protein, composed of three subunits

Associated with the bacterial RC from

Rh. sphaeroides are the following

cofactors: four bacteriochlorophylls, two bacteriopheophytins, two ubi quinone molecules, and one nonheme Fe2+. The arrangement of these cofac tors

(35, 36) in the RC from Rh. sphaeroides is shown in Figure 2. Two

closely associated bacteriochlorophyll molecules near the periplasmic side of the

RC form a bacteriochlorophyll dimer that serves as the primary electron (D). The other cofactors, bacteriochlorophyll (B), bacteriophenophytin

donor

865


\ '-......

CH3


\ 2.6 . l-J---P= 5.3 l...cP r-IAr- gL - 2- 17I \�}i� 3.5\ /4.5 P N

-

•

.. H' 2 ' 45'.

�---,'

•

-N�

2.!.�.··:, ....f ...

-Q'

» 5.2\ :'

CH3 CH3 3-8-- -'0" .3 7 ,I IAsp L213 1 6.8 C -O----- ----------·

·

,

"

.

. ,'

:

.

0

,-

..

.

\ -C

.

"

Fe

O

'1-'-1---- -- ----- 8.7· ----i�,u L� 21'

I Asp L210 Ic =0 /

Figure 3

IHiS L1901

�Q

� ISerL2231

-

Schematic representation of the structure near the QB- bindin g site. The closest

\ Modified distances (in angstroms) between neighboring residues are indicated.

from reference 38

with p ermis sion .

: 5 3 (A-QA � cf>AQA-,

3.

the electron transfer rate, k'hQA' is 5 X 1 09 S-1 and is relatively independent of temperature and pH (27). The proton uptake rate associated with this reaction was found to be much lower, 104 S-1 (pH 6). The rate depends on pH, decreasing at higher pH with a slope d(log k)/d(pH) - 0.3 (15). From the temperature dependence of the rate, an activation energy of >40 kllmol (0.4 eV) was found (32). Takahashi et al have explained the proton uptake rate, the relatively high activation energy, and the low viscosity dependence for the reaction in terms of a collisional reaction between pH indicator molecules and proton acceptor groups that reside on the protein and whose accessibility may be determined by conformational equilibria of the protein (32). Supporting evidence for a conformational change at this step is the observation by Brzezinski et al. (67) of an electrogenic event upon reduction of QA to QA that has the opposite sign from that observed for proton uptake. This electrogenic event was suggested to be due to a conformational change upon QA reduction. The reduction of QA - to the doubly reduced QA 2- state occurs much more slowly (10 ms) (68) than the first reduction (200 ps) and is observed only under extreme conditions of high illumination. The reduced rate of the second electron transfer to QA may be related to the absence of an efficient proton transport chain to QA from the solvent. The double reduction of QA is enhanced in Res from which Fe2+ has been removed. This is probably due to an increased proton accessibility of the QA site (69), brought about by =

-

874

OKAMURA & FEHER

possible structural changes associated with the removal of Fe2+. In these RCs the electron transfer rate k'hQA is lowered considerably (70). Thus, there seems to be a correlation between fast electron transfer and inaccessibility to protons (see footnote 1). The rates of proton transfer and electron transfer due to the reaction


k�� QA-QB� QAQB-

4.

were found to be very similar. Both the electron transfer and proton transfer rates were about 1 04 S-1 at low pH and decreased with increasing pH. The reason for the coincidence between these rates may be that proton transfer is limited by electron transfer (or electron transfer is limited by proton transfer) . In RCs from Rh. sphaeroides different values for k(;:B have been found by different workers. Wraight (15) and Vermeglio & Clayton (47) found a relatively constant decrease in the log of the rate with increasing pH with a slope [d(log k)/d(pH)] of about - 0.3. Kleinfeld et al (48) reported a pH independent rate at low pH that decreases above pH 9. Takahashi et al (32) have reported that the rates are biphasic, with the fast phase agreeing with the results of Kleinfeld but with an additional slower phase. The decrease in k(;:B with increasing pH is consistent with the presence of an acidic residue near QB that must be protonated before electron transfer can occur ( 15). The data of Kleinfeld et al could be fitted to a simple model in which the protonation state of a residue with pKa of ca. 9 determined the rate of electron transfer (48). This residue was shown by site-directed mutagenesis to be GluL212 (7, 7 1 ) . In mutant RCs containing GIn in place of Glu at position L212 the rate of electron transfer remained constant at high pH, showing the absence of an interacting, titratable residue. At high pH, the negatively charged GluL21 2 must become protonated before fast electron transfer can occur. Thus, the kinetics of electron transfer can give information about the rates of proton transfer to GluL212. The kinetics of electron transfer seen by Kleinfeld et al (48) at higher pH are monophasic, suggesting that the rates of protonation and deprotonation of GluL21 2 are high compared with those of electron transfer; e . g. at pH 9 the proton transfer rate should be higher than 1 04 s - 1. The biphasic kinetics for Jc104 S-I; pH 9) and a low rate (200 S-I; pH 9) of proton transfer. The differences between the results seen by Kleinfeld et al (48) and Wraight ( 15) are not understood at present but may be due to differences in sample preparation or detergent. Biphasic rates of other reac tions have also been observed: for k(J!B in RCs from a mutant in which AspL21 3 was changed to Asn (72), for kAD in RCs in which ubiquinone was replaced by anthraquinone (73), and in the recombination reactions of R . viridis (74). The proton uptake rate associated with the second electron transfer reac tion, 5. has been found to be similar to the electron transfer rate k(;:B (15, 32, 53). Both the electron transfer and proton uptake rates associated with the second electron k500 350 300

k�� (S-I) 1 300 750 >500 4 �1O >1000 >500 0.4

kH

(slow)

(S-I)

1 200 6 >500 4

0.3

Turnover (cytlRC) (S-I) >500 12 �200 8 �1O > 1 50 > 1 50 0.8 0.6

AspL2 1 0

�

Asn

800

ArgL217

�

Gin

4000

ArgL2 1 7

�

Leu

3200

> 1 50

HisLl90

�Gln

1 500

200

AspL2 1 3

�Asn,

GluL2 1 2

�Gln

4000

290

600 >200

0.4

>200

0.6

Ref. 7, 19 7, 71 76 19 74a 19 74a 20,78 M. Paddock, unpublished M. Paddock, unpublished M. Paddock, unpublished M. Paddock, unpublished J. Williams, unpublished 71; M. Paddock, unpublished

in Figure 4). When proton transfer is the rate-limiting step in the quinone reduction cycle, the rate of cytochrome turnover equals twice the proton transfer rate since two cytochrome molecules are oxidized in one cycle. A reduced proton uptake rate kH can result from a mutation that changes the pathway to QB. A slow protonation would be observed when the proton uptake associated with the second electron transfer is measured either after 2 single turnover flashes « 1 p,s duration) or after a single multiturnover flash (> 1 ms duration). In these cases the reaction center would be rapidly driven to the rate-limiting proton uptake step (of either H+(1) or H+(2), see Figure 4). A slow rate of electron transfer would occur in a mutant in which proton uptake was inhibited and in which proton uptake was required before electron transfer. Thus, in the model shown in Figure 4 transfer of the second electron would be slow if H+(1) were blocked but would be unaffected if H+(2) were blocked. (The state DQAQ�- in Figure 4 is expected to be a transient intermediate state with a low occupancy.) A list of mutants constructed to study proton transfer is shown in Table 1. In the first proton uptake mutant constructed, GluL212, an acidic group close to the QB site, was changed to GIn by Paddock et al (7). The cytochrome turnover rate in this mutant was slowed by a factor of >40 following the fast oxidation of three cytochromes (Figure 6). This can be explained by a bottleneck in the transfer of the second proton, H+(2) (see step 5 in Figure 4),


�

0 0::

----

� Light off

.f Lig ht on

0

877

>-.-

0

a to 10

Z-.

0

,

Figure 6

0.5

60

Cytochrome turnover rates in native and mutant RCs. RCs were illuminated in the

presence of excess cytochrome c and

QIO'

The mutants SerL233

--+

Ala and GluL212

--+

Gin

show a fast oxidation of two and three cytochromes, respectively, followed by a reduced cytochrome oxidation rate that indicates a bottleneck in the proton transfer rate. Modified from references 7 and 19.

which slows the turnover of the quinone reduction cycle. Since this block occurs after electron transfer of the second electron, the RC can oxidize another cytochrome (three altogether) to form the fully reduced quinone complex QA-(QBH)- . This suggested that electron transfer of the first and second electrons is unimpeded and that the rate limitation is due to a bot tleneck in proton transfer (7). This proposal was verified by direct measure ments of the electron and proton transfer rates that showed that electron transfer rates for both k Gin mutants were obtained by Takahashi & Wraight (7 1 ) .

and > 1 1 , for the other (Table 2). Tentative assignments of the groups responsible for the pH dependence of the rates have been made by studying Res modified by site-directed mutagenesis. The pH dependence above pH 9.3 in native Res is eliminated in Res from the GluLZI Z -7 GIn mutant (7, 71). This is consistent with the assignment of a pKa of 9.3 to GluL2I 2. In Res in which AspLZ13 (3Z, 71, 76) and

PROTON TRANSFER IN REACTION CENTERS Table 2

pK.s for acidic residues obtained from kSD measurements·

RC


Native GluL2 1 2 GluL2 1 2 AspL2 1 3 AspL2 10

883

-.>

-.>

-.>

-'>

pK , (Qs)

pK , (Qs-)

pK2 (Qs)

pK2 (Qs-)

S . Sb S . Sb

6. 1 6.4

9.3

>11

8 . 3b

>10 >9

Gin Asp Asn Asn

4.7

6.4

7.8

' These pK. values were obtained by using EIj. 7 and 8

Res containing QB or QB-'

for residues labeled 1 or 2 in

b This represents a maximum value. If the pH-independent region is due to

back reaction

(7 1), the

actual

pK,

a direct

would be lower.

AspL210 were replaced by Asn (77), the transition at 9 . 3 is shifted to lower pH (8.3 and 7.8 for AspL213 and AspL210, respectively). Takahashi & Wraight (71) have suggested that the pH-independent region in the AspL213 � Asn mutant is due to the direct electron transfer from D+QAQB - to DQAQB ' (In this case the pKa for GluL212 in this mutant may be at a lower pH.) In the double mutant with AspL213 -+ Asn and GluL212 -+ GIn the value of kBO is ca. 0.1 S-1 and is pH independent (71), showing that the pH dependence in the single AspL213 -+ Asn mutation was due to the shifted pKa of Glu-L212. The changes in pKa can be understood in terms of electrostatic interactions between titrating residues (for a good discussion see reference 84). The shift in the pKa of GluL212 from 9.3 to 7.8 in the AspL210 mutant indicates an interaction between GluL212 and AspL210 of ca. 1.5 pH units (90 meV). From this interaction an effective dielectric constant of ca. 1 6 can be es timated if the energy is calculated by using Coulomb's law and the known distance of 9. 8 A (average distance between carboxyl oxygens). The electrostatic interactions between the two Asp residues and GluL212 is partially responsible for the apparent high pKa of 9 .3 for GluL212. The two Asp residues must have intrinsic pKas (owing to interaction with ArgL21 7, and possibly more effective dielectric screening) lower than that of GluL212 and hence ionize at a lower pH. The pKa of GluL212 is raised by the sum of the electrostatic interactions with the Asp residues (ca. 3 to 5 pH units). In RCs from the GluL212 -+ Asp mutant the high-pH transition changes from pKa of 9.3 to pKa of < 5 . This large change in the apparent pKa of an acidic residue as a result of the removal of a methylene group is unusual. However, it may be explained if the mutation results in a change in the order of ionization of strongly interacting residues; i.e. the intrinsic pKa of AspL212 has been lowered below the intrinsic pKas of AspL21 0 and AspL213. The pH dependence in the region from pH 5 to 7 in native RCs was eliminated in both the AspL213 -+ Asn (20, 83) and AspL210 -+ Asn (77)


884

OKAMURA & FEHER

mutants. This may be explained if in these mutants the pKa of the remaining carboxylic acid residue besides GluL2 1 2 (AspL2 1O or AspL2 1 3) was shifted to lower values (ca. 3). Electrostatic calculations have indicated that the AspL2 1 3 has a lower pKa(QB) « 4) than AspL2 10 (P. Beroza, unpublished data) owing to interaction with ArgL2 1 7 . The pKa(QB) of AspL2 10 is then assigned a value of ca. 5 . The pKa(QB -) values of both AspL2 10 and AspL2 1 3 are estimated to be similar and are consistent with the value of ca. 6 observed experimentally (Figure 8; Table 2) . It seems, therefore, that both are implicated in the 5 < pH < 7 dependence of kAB . Why, then, is the pH dependence removed when either of the two Asp residues is mutated? An explanation lies in the interaction between the two acidic groups; i.e. in the mutant the remaining carboxylic acid residue is shifted to a lower pKa value (below the observed range) owing to loss of interaction with the acidic group that has been removed. Another way to obtain information about proton and electron transfer equilibria is to measure the pH dependence of the midpoint potential (Em) for quinone reduction in the RC . The Em for the reduction of QA has been found by many workers to decrease by 60 mV per pH unit, as expected for a reduction in which one proton is taken up per electron (41). This pH depen dence is inconsistent with the measured proton uptake of less than one proton per electron discussed earlier. A possible reason for this discrepancy is the difference in the time scale of the measurements of midpoint potential and proton uptake or the difficulty in achieving redox equilibrium between the RC and the monitoring electrodes . An indirect method of measuring the pH dependence of the free energy change due to QA - formation, based on the intensity of delayed fluorescence, agrees with the lower values for proton uptake (85).

Electrogenicity The proton transfer events associated with quinone reduction have also been studied by measuring the photoinduced voltage across membranes (e.g. lipid monolayers or bilayers) containing oriented RCs (86-90) . This photo voltage (electrogenicity) is a measure of the electrical work performed in going from one state of the RC to another and is related to the dielectric ally weighted charge displacement across the membrane. The electron transfer k 8 the electron transfer was electrogenic, indicating a charge displacement perpendicular to the membrane. The displacements were interpreted to arise from proton uptake by acidic residues driven by pKa shifts due to QB - formation. The pH dependence of the amplitudes of the displace-



885

ment currents agreed with the pK shifts expected from the pH dependence of the back-reaction kinetics, kBO' The electrogenicity associated with the second electron transfer reaction, k(�B' which occurred in oriented RCs after a second flash, had a characteristic time of ca. 1 ms (pH 7.5) (89, 9 1). This electrogenicity is most probably due to proton transfer since electron transfer is parallel to the membrane. The amplitude of the electrogenic signal associated with this proton uptake de creased approximately by one-half (pH 7) in the GluL212 � GIn mutant (93). This result supports the model in which GluL212 is responsible for the transport of one of the two protons taken up in this step.

MECHANISM OF PROTON TRANSFER Proton transfer in bacterial Res is a complex phenomenon, whose details have so far not been worked out in final form. We shall summarize here the present state of knowledge about four aspects of the reaction mechanism: (a) the sequence of electron and proton transfer steps, (b) the pathway of proton transport, (c) the dynamic model of proton transport, and (d) the energetics of proton transport. Sequence of Electron and Proton Transfer Steps

The direct protonation of QB is associated with the transfer of the second electron to QB as shown by spectroscopic and proton uptakc measurements. The proposed mechanism for this step involves thc uptake of two protons, H+(l ) and H+(2). The first proton uptake step could occur either before or after electron transfer as shown below (also see Figure 4) (53):

9.

The experimental data are consistent with either mechanism. The upper path (proton transfer before electron transfer) would be expected to dominate if QAQB2- is higher in energy than QAQBH; the lower path (electron transfer before proton transfer) would dominate if QAQBH is higher in energy than QAQB2- . A difficulty in deciding experimentally between the two mech anisms is that both QAQBH and QAQB - are high-energy intermediates that are not formed in sufficient quantity to be observed. Furthermore, we do not know from experimental measurements whether electron transfer precedes or follows proton transfer, since these individual steps have not yet been re-

886

OKAMURA & FEHER

solved kinetically. Thus, in the absence of reliable calculations of the energies of the intermediate states, we can only speculate under what conditions one or the other pathway would dominate. The energy required to reduce QB - to QB2- (without protonation) depends on the environment of the quinone and is higher in aprotic solvents than in aqueous solutions. For instance, for ubiquinone the midpoint potential Em between Q- and Q2- (unprotonated) is at 1 15 V in acetonitrile (28) but can be estimated to be about - 0.3 V in aqueous ethanol [calculated by assuming Em(2) 0.45 V , pKa(2) 13 , and pKa(2) , 1 4]. If the Em for QB2- in Res is close to that in acetonitrile, the proton transfer (upper) path should domi nate. However, if Em is similar to that in aqueous ethanol, the reaction (lower path) could have an activation energy less than 300 mY, making it favorable for electron transfer to precede proton transfer. The energy required to form the protonated semiquinone state QH depends on the difference between the pH and the pKa. The protonated semiquinone state has never been observed by optical spectroscopy even at low pH (down to pH 5) despite the fact that there are negatively charged residues, e.g. AspL213, near QB. Thus its pKa might be very low « 4). In this case the reduced QB and QB2- states may be stabilized with respect to the protonated state, for instance by hydrogen bonding, permitting the electron transfer to proceed before protonation. However, an alternative explanation for the lack of observation of the protonated semiquinone invokes interactions with near by acid residues? These interactions could lower the energy needed to protonate the semiquinone at pH 7 to a low value (e.g. 1 pH unit, i.e. 60 meV) and proton transfer could precede electron transfer. It is also possible that proton transfer and electron transfer are highly cooperative and closely coupled. In this case electron transfer and proton transfer may proceed simultaneously.


-

=

=

.

=

-

Structural Models for the Proton Transfer Pathway

A structural model for the proton transfer pathway in Res from Rh. sphaeroides has been based on site-directed mutagenesis studies in which the residues that significantly affect proton transfer were identified (19, 71) (see above) and on inspection of the X-ray crystal structure of the Re. This model is shown in Figure 9. In this model two protons, H+(I) and H+(2), are transferred from the external solvent to QB along two pathways. The first proton, H+( l ) , is transferred along a pathway involving AspL213 and SerL223. The second proton is transferred along a pathway involving AspL213 and GluL212. Inspection of the X-ray crystal structure of the protein shows that protons from solution can find access to the region near AspL210 2The lack of observation of the protonated semiquinone at lower pH may be due to a pH-dependent pK. in which the pKa for the protonated semiquinone decreases at lower pH owing to the protonation of nearby acid groups, e.g. AspL2 1 3 .



887

Figure 9 Proposed pathways for proton transfer in Res from Rh. sphaeroides. Protons from the aqueous phase (hatched area) can approach AspL2 l 3 via aqueous channels in the protein. The first proton , H + ( l ) (solid line), taken up by the quinone carbonyl H·bonded to SerL223, was shown to transfer via a pathway involving AspL2 1 3 and SerL22 3 . The second proton, H + (2) (dashed line), taken up by the carbonyl H·bonded to HisL l 90, was shown to transfer via a pathway involving GluL2 l 2 and possibly AspL2 1 3 . A cavity (shaded) near the methoxy groups of QB, presumably containing internal water, is likely to play a role in the proton transfer.

or ArgL217, although evidence for the direct involvement of these residues is lacking (Table 1 ) . In addition, a void in the protein structure large enough to accommodate five or six water molecules can be visualized in the X-ray crystal structure in a region bordered by the methoxy groups from QB , AspL21 3, and GluL21 2 (P. Beroza, unpublished results). This pocket is likely to contain disordered water molecules, which undoubtedly play an important role in the proton transfer to QB ' Bound water molecules have been observed in this region in the X-ray crystal structure of the RC from Rps. viridis (25). The large changes in proton transfer rates that accompany the structurally conservative mutations of proton donor residues suggest that proton transport in the Rh. sphaeroides RC is a specific process involving defined pathways (at least for residues close to the quinone). This would be expected since proton transfer can occur only if appropriate donor and acceptor groups are posi tioned within the proton transfer distance of a few angstroms of each other (94). The difficulty in observing changes in proton transfer rates as a result of mutations of residues farther away from the quinone site (e. g. AspL21O, ArgL21 7) may be due to multiple alternative parallel pathways that circum vent these residues. The pathway shown in Figure 9 is based on the assumption that the large changes in proton transfer rate due to mutation of GluL212, SerL223, and


888

OKAMURA & FEHER

AspL213 result from local changes in the proton accessibility near the mu tated residue and not from conformational changes. In addition, the model makes the simplest assumption, i.e. that the loss of activity due to mutation of a protonatable residue indicates its role as a proton donor in a proton transfer chain. Other interpretations of the results from site-directed mutagenesis are possible; these include (a) short-range steric effects or long-range con formational effects not related to the role of the mutated residue as a proton donor and (b) electrostatic effects on either the rate or the equilibrium in the proton transfer reaction. Mechanisms for proton transfer consistent with the observed changes are discussed in the next section. There is evidence that in RCs from other bacterial species different path ways are operative. In RCs from Rps. viridis and Rsp. rubrum AspL213 is not conserved but is changed to Asn (95). However, these RCs have a compensat ing charge modification. The residue AsnM44 close to QB in Rb. sphaeroides is found to be changed to Asp at the homologous M43 position in RCs from both Rps. viridis and Rsp. rubrum . Recently a double mutant containing the AspL21 3 � Asn and Asn M44 � Asp mutations was constructed (S. Rongey, unpublished results). The second mutation (AsnM44 � Asp) re stored high turnover and proton transfer rates and photosynthetic competence that was lost in the AspL213 � Asn single mutant. This supports the idea that proton transfer in Rps. viridis and Rps. rubrum involves AspM44. Thus, the proton transfer pathways may be quite different in different RCs. The RC of photosystem II (PSII) from oxygenic photosynthetic organisms contains a QB site with properties similar to those in the RC from purple bacteria (reviewed in reference 96). In the PSII RCs from Aramanthus hybridus, mutation of the residue Ser264 on the DI subunit (homologous to the SerL223 in Rb . sphaeroides) to Gly does not alter the rate k(;':B (97), as it does in the RC from Rb. sphaeroides. This result may be due to a different pathway for proton transfer to QB in PSII RCs. Another interpretation is that a water molecule substitutes for the Ser OH group in the Gly-containing mutant. Takahashi & Wraight (79) have found that weak acids such as azide will facilitate the transfer of protons to QR in RCs from Rh. sphaeroides from mutants whose proton transfer rates were blocked. These workers have proposed that bicarbonate plays a similar role in RCs from PSII. This proposal would explain many of the important regulatory effects of bicarbonate on the functioning of the PSII RC (98). Thus, bicarbonate regulates proton transfer, which in tum may regulate photosynthetic growth of plants. A model in which bicarbonate binds to the nonheme iron in the PSII RC has been discussed by Diner et al (96). Dynamic Models of Proton Transfer

A mechanism of proton transfer must ultimately describe the molecular events. that lead to proton transport. Several molecular mechanisms for proton trans-



889

fer in proteins have been discussed (99-10 I ) . One model involves proton transfer through a hydrogen-bonded chain of proton donor residues (99). An examination of the RC structure from Rh. sphaeroides did not reveal a hydrogen-bonded chain between protein residues from the exterior of the protein to QB ' However, a hydrogen-bonded chain could be present if bound water molecules that are unresolved in the X-ray crystal structure are present. For instance, the proposed proton donor residue to GluL212 is AspL21 3 at a distance of 7 A. There is a void between these two residues that probably contains water molecules which could form hydrogen bridges between the residues. Thus, water molecules undoubtedly play an important role in transporting protons to QB ' Several different dynamic models for proton transfer may be invoked to explain the results obtained by site-directed mutagenesis. These are shown on the left of Figure 10 for the transfer of the first proton and involve SerL223 and AspL2 1 3 . The series of steps 1 , 2, and 3 (sequential mechanism) involve

the sequential protonation of AspL213, SerL223, and QB -. A similar mech anism (concerted mechanism) involves steps 1 and 4, in which proton transfer from AspL213 and SerL223 occurs in a concerted manner [a similar mech anism, proposed for serine proteases, has been extensively investigated (102)]. Whether the sequential or concerted mechanism dominates depends on the energies of the charged states . Steps 1 and 5 constitute a direct mechanism involving the direct transfer of a proton from AspL213 to QB -. In this mechanism the reaction product, the protonated quinone, initially has a different structure, since the transferred proton does not come from the hydrogen bond with the SerL223 . Takahashi & Wraight favor the direct mechanism (71), in which case the effects of the Ser - Ala mutation at L223 is attributed to a steric effect. This assignment does not explain the effects of the Ser - Thr, Ser - Asp, and Ser - Asn mutations in which only the mutants with protontable residues exhibit normal proton transfer although all may have similar steric effects. Another, and possibly the most general, mechanism (hydronium ion mechanism) involves steps 6 and 7 (or 6, 8, and 3), in which a protonated internal water molecule, H30+, acts as the proton donor group and the role of AspL213 (negatively charged) is to stabilize the positively charged hydronium ion. The hydronium ion mechanism would explain the proton transport in Rsp . rubrum and Rps . viridis RCs, and in the double mutant (AspL213 - Asn, AsnM44 - Asp) in Rh. sphaeroides RCs, where the residue at AspL213 is replaced by Asn with a compensating modification of Asn to Asp at the position homologous to M44 in Rh. sphaeroides. This modification conserves the electrostatic stabilizing proper ties of a negatively charged carboxyl group without having to invoke an entirely new pathway for proton transfer. Several mechanisms for proton transport of the second proton are shown on the right of Figure 10. The doubly reduced, singly protonated quinone can


H+

(1 )

H+ (2)

'\ Ser L223

o O-H 0.:.0-0 ?

H2 0 0 / =Asp L213

-

\

(y

0.. H 0-0-0 (i;) )5 H2 0 =o ,? (2)

'\ + @H ?

,?=

_

o

0-0 -0 H20

_

Gp

_

,? =O

Figure 10

H-O-Q-O H 20

H

�o

Glu

\

rHlO+ ?- v

�, .. o- � O n

/=0

L21 2 /c?=o

l�

(3P+

(41

)-l H/O-o- O

Fe � h

- N

G H2 0

)=0

(10�

,h3)

?,? =O

/=0 _

Possible mechanisms for the transfer of the first proton, H+( l ) (left), to QB

H.,N

�

H."N� Fe W-0-Q- � H20

\O-H .r:>P-� ,=/", 0 � 0 H 2 ?

represents the proton originating in the outside solvent,

� Fe

p ,?= O

e

o H20

� (11 1

\..H 0':0-0

(3) \

e-

DB

�

H,s l1 90 /o -�- H ."NV ( QB H I-

-

and the second proton, H+(2)

(right), to (QBH) - . The circled H



891

accept a proton by different mechanisms involving GluL212. The proton can be transferred from GluL212 to the quinone directly (step 11). Alternatively, a protonated water molecule may be involved (steps 9 and 10). Another mechanism might involve the stabilization of the QB2- state by deprotonation and reprotonation of HisLl 90 (steps 12 and 13) (this proton transfer might also occur before electron transfer). After protonation, the doubly reduced quinone dissociates from the QB-binding site and is replaced by an oxidized quinone molecule (50). The driving force for the dissociation of QBH2 is the lowered binding affinity of the dihydroquinone, probably as a result of the loss of H-bonds from the amino acids. Energetics of Proton Transport

In the previous section we discussed different mechanisms of proton transfer. Which of these mechanisms dominates will depend critically on the energy of the different states involved in the process ( 1 01). Thus, an understanding of the energetics is of the utmost importance. A critical point in the energetics is the calculation of the energy required for transferring charges inside proteins (102-104). These calculations present a difficult task since they require the calculation of small differences between large energies. An additional com plication is the electrostatic interaction between the many charged residues of the protein (66). Work in this area is currently in progress in several labora tories, and the results will be important for the full understanding of the mechanism of proton transfer. In the absence of accurate calculations, a tentative working model for the energetics of proton transfer for the sequential mechanism based on ex-

I

proton

·5

0 5

energy ' 10 ( pKa ) 15

H 0+ �

'\ H

R OH 2+

eOOH Asp L21 0

+

(1 )

+

H (2)

____ _ _

- - -

�

eOOH

/ Ser L223 ""

Asp L21 3' "

,

or

H2O

QSH

"- eOOH Glu

L21 2" "-. Q S H 2

20 Figure 1 1 Estimated energies of the protonated donor groups in the RC from R. sphaeroides that

are thought to be involved in the transfer of H + ( 1 ) (solid arrows) and H+(2) (dashed arrows). The

pKa * levels in the diagram represent energy levels of the protonated states in the proton transfer pathway for a single proton as it proceeds from solution to a QB - molecule with a single negative charge [either QB - or (QBH) -] at pH

7. At pH 7 the states with pKa *s less than 7 will be

unprotonated and those with pKa*5 greater than 7 will be protonated.


892

OKAMURA & FEHER

perimental results and rough estimates is presented in Figure 11. It shows the energy levels (pKa* ) of the different groups proposed to be involved in the transfer of the first and second proton. The pKa* values are the energies required to protonate residues in the RCs when the protonation states of the acidic residues are in equilibrium at pH 7 and QB has a single negative charge, i.e. the state of the RC during the uptake of the first and second proton, assuming that proton transfer occurs before the second electron transfer. The pKa * values will generally not be the pKas that would be measured by titration since the protonation state of interacting residues would change. The es timates are based on interaction energies of 2 to 4 pH units for nearby charged residues calculated from Coulomb's Law and assuming an effective dielectric constant of 10 to 20. The value of the intrinsic pKas were those in aqueous solution. No attempt was made to account for the change in the intrinsic pKa value due to the protein environment. The pKa* for the protonated semiquinone QBH in an environment contain ing several negative charges (AspL213, AspL210) is partially compensated by the positive charges of ArgL217 and Fe2+. Its value is estimated to be ca. 6, increased by ca. 1 pH unit over the value in aqueous solution (39) but less than 7 to explain the lack of observation of the protonated semiquinone by optical spectroscopy at pH 7. The pKa* for the protonated Ser hydroxyl group ROH2+ is estimated to be ca. 2 from the solution value of - 2 ( l 05) shifted by about +4 pK units, mainly owing to strong interactions with the nearby negative charges on QB and AspL2 1 3 . A hydronium ion (H30+ ) in the same region would have approximately the same pKa. The pKa* for the carboxyl group of AspL21 3 is estimated to have a value of ca. 6 owing to interactions with ArgL217, Asp21O, and QB - . The values for protonated Ser, the hydro nium ion, and AspL213 are all close enough to that of the protonated semiquinone to indicate that they can serve as efficient proton donors to QB -. In contrast, the pKa* of the carboxyl group of GluL2l 2 (when QB is negative ly charged) is ca. 12, which may be too high for it to serve as an efficient proton donor to QB -. This may explain why GluL212 does not replace SerL223 as the donor of the first proton in the SerL223 � Ala mutant. The pKas of the neutral Ser hydroxyl group and water molecule are ca. 15 in solution (ca. 19 in the region near QB -), making proton transfer to QB - by these neutral species unlikely. The pKa* of the carboxyl group on AspL210 is estimated from the pH dependence of kBD to be ca. 6. The proton transfer chain for the second proton is shown in the lower path of Figure 11. The pKa* of the dihydroquinone QBH2 is ca. 12 in aqueous solution (28); the value in the RC should be somewhat higher, ca. 13, again, increased by ca. 1 as was the pKa* of the semiquinone3 (see above). This 3>fhe pK.* for QaH2 after proton transfer should also be increased by interaction with th(: ionized GluL2 l 2 if this group is its immediate proton donor. However, the ionized GluL2 12 should become rapidly reprotonated.



893

large change in pKa upon transfer of the second electron is the driving force for the uptake of both protons. The proton transfer path is proposed to be the same as that for the first proton up to the branch point where a proton is transferred to GluL2l 2. The pKa of the carboxyl group of GluL2l 2 (in the presence of a single charge on QB) of 12 makes GluL21 2 a good donor to QBH- but not to QB -. Although the estimates are very rough and will likely require revision, they serve as the basis for discussion and further ex perimentation. The energetics of proton transfer appear to avoid large in creases in the free energy of proton transfer between members of the chain. For a proton to be transported to QB - it must surmount a barrier imposed by the protonation of groups with low pKas, e.g. Ser, hydroxyl group, or internal water. Although the energies of the protonated states of these groups are lowered by negative charges, there still remains an activation barrier of approximately 4 pH units (240 meV), assuming that a proton donor with a pKa* of 6 (e.g. AspL21O) is involved in the chain. This barrier is not unreasonably high and should appear as an activation energy for proton transfer.

SUMMARY Proton transfer in the bacterial RC associated with the reduction of the bound QB to the dihydroquinone is an important step in the energetics of photosynthetic bacteria. The binding of two protons by the quinone is associ ated with the transfer of the second electron to QB at a rate of ca. 1 03 s - 1 (pH 7). Mutation of three protonatable residues, GluL212, SerL223, and AspL21 3 , located near QB to nonprotonatable residues (GIn, Ala, and Asn, respectively) resulted in large reductions (by 2 to 3 orders of magnitude) in the rate or proton transfer to QB. These mutations can be grouped into two classes: those that blocked both proton transfer and electron transfer (SerL223, and AspL2l 3) and those that blocked only proton transfer (GluL212). These results were interpreted in terms of a pathway for proton transport in which uptake of the first proton, required for the transfer of the second electron, occurs through a pathway involving AspL21 3 and SerL223. Uptake of the second proton, which follows electron transfer, occurs through a pathway involving GluL2l 2 and possibly AspL213. Acidic residues near QB affect electron transfer rates via electrostatic interactions. One residue, with a pKa of ca. 10 interacting strongly with the charge on QB (LlpKa > 2), was shown to be GluL2l 2. A second residue with a pKa of ca. 6, which interacts more weakly with the charge on QB (LlpK "" 1 ) , could be either AspL210 or AspL21 3. Several possible mechanisms for proton transfer are consistent with the observed experimental results and proposed proton pathways. These involve proton transfers from individual amino acid residues or internal water mole-

894

OKAMURA & FEHER

cules either as single steps or in a concerted fashion. The determination of the dominant mechanism will require evaluation of the energetics of

steps.

the various

ACKNOWLEDGMENTS

We thank our collaborators, especially Paul McPherson

and Mark Paddock,


whose PhD theses formed the basis for much of this work; Paul Beroza , Peter

Brzezinski, Art Chirino, Don Fredkin, Adam Messinger, Doug Rees, Scott Rongey, and JoAnn Williams for helpful discussions and permission to cite their unpublished work; and Colin Wright for sending copies of manuscripts prior to publication. The work from our laboratory was supported by grants from

NSF and NIH.

Literature Cited 1 . Feher, G . , Allen, J. P . , Okamura, M. Y. , Rees, D. C. 1 989. Nature (London) 339: 1 1 1-16 2. Breton, J . , Verrneglio, A. 1988. The Photosynthetic Bacterial Reaction Cen ter: Structure and Dynamics. New York: Plenum. 443 pp. 3 . Norris, J. R . , Schiffer, M. 1990. Chem. Eng. News 68(3 1 ):22-28 4. Michel-Beyerle, M. E . , ed. 1990. Reac tion Centers of Photosynthetic Bacteria . New York: Springer-Verlag. 469 pp. 5 . Mitchell, P. 1 96 1 . Nature (London) 1 9 1 : 1 44-48 6. Cramer, W. A., Knaff, D. B. 1990. Energy Transduction in Biological Membranes. New York: Springer Verlag. 545 pp. 7. Paddock, M. L . , Rongey, S. H . , Feher, G. , Okamura, M. Y. 1989. Proc. Natl. Acad. Sci. USA 86:6602-6 8. Baltscheffsky, H. , von Stedingk, L.-V. 1966. In Currents in Photosynthesis, ed. J. B. Thomas, J. C. Goedheer, pp. 2536 1 . Rotterdam: Ad. Donker. 9. Cogdell, R. J . , Jackson, J. B. , Crofts, A. R . 1973. J. Bioenerg. 4:21 1-27 10. Halsey, Y. D . , Parson, W. W. 1974. Biochim. Biophys. Acta 347:404-16 1 1 . Petty, K. M. , Dutton, P. L . 1 976. Arch. Biochem. Biophys. 172:335-45 1 2 . Reed, D. W. , Clayton, R. K. 1968. Biochem. Biophys. Res. Commun. 30 : 47 1-75 1 3 . Feher, G. 1 97 1 . Photochem. Photobiol. 1 4:373-87 1 4 . Feher, G . , Okamura, M. Y. 1978. See Ref. 108, pp. 349-86 1 5 . Wraight, C. A. 1979. Biochem. Bio phys. Acta. 548:309-27 16. Deisenhofer, J . , Epp, 0. , Miki, K. , Hu-

er, R . , Michel, H. 1984.

J. Mol. Bioi.

1 80:385-98

1 7 . Allen, J. P. , Feher, G . , Yeates, T. 0. , Rees, D. C. , Deisenhofer, J . , et al.

1986. Proc. Natl. Acad. 83:8589-93

Sci.

USA

1 8 . Chang, C.-H. , Tiede, D., Tang, J. , Smith, U. , Norris, J. , Schiffer, M. 1986. FEBS Lett. 205:82-86 1 9 . Paddock, M. L . , McPherson, P. H . , Feher, G . , Okamura , M . Y. 1 990. Proc. Natl. Acad. Sci. USA 87:6803-7 20. Takahashi, E. , Wraight, C. A. 1990. Biochim. Biophys. Acta 1020: 1O7- 1 1 2 1 . Khorana, H . G . 1988 . J . Bioi. Chem. 263:7439-42 22. Senior, A. E. 1990. Annu. Rev. Bio phys . Biophys. Chern. 19:7-41 23. Kaback, H . R . 1990. Philos. Trans. R. Soc. London Ser. B 326:425-36 24. Malmstrom, B. G. 1 989. FEBS Lett. 250:9-21 25. Deisenhofer, J., Michel, H. 1989. EMBO J. 8:2149-70 26. Rees, D. C. , Komiya, H. , Yeates, T. 0. , Allen, J . P. , Feher, G. 1989. Annu. Rev. Biochern. 58:607-33 27. Kirrnaier, C. , Holten, D. 1987. Photo synth. Res. 1 3:225-60 28. Morrison, L. E. , Schelhorn, J. E. , Cot

ton, T. M. , Bering, C. L. , Loach, P. A. 1 982. In Function of Quinones in Ener gy Conserving Systems, ed. B. L. Trum power, pp. 35-58. New York: Academ ic 29. Williams, R. J. P. 1988. Annu. Rev.

Biophys. Biophys. Chem. 17:7 1-97 30. Copeland, R. A. , Chan, S. 1. 1989. Annu. Rev. Phys. Chem. 40:671-98 3 1 . Feher, G. , McPherson, P. H. , Paddock,

M. , Rongey, S. , SchOnfeld, M. , Oka-



mura, M. Y. 1990. See Ref. 107, pp. 1. 1 . 39-46 32. Takahashi, E . , Maroti, P. , Wraight, C. A . 199 1 . In Electron and Proton Trans fer in Chemistry and Biology, ed. E . Diemann, W. Junge, A . Muller, H . Ratajczaks. Amsterdam: Elsevier. In press 3 3 . Allen, J. P . , Feher, G . , Yeates, T. 0 . , Komiya, H . , Rees, D . C . 1987. Proc . Natl. Acad. Sci. USA 84:61 62-66 34. Chang , C.-H. , EI-Kabbani, 0 . , Tiede, D . , Norris, J . , Schiffer, M. 199 1 . Biochemistry 30:5352-60 3 5 . Allen, J. P . , Feher, G . , Yeates, T. 0 . , Komiya, H . , Rees, D . C. 1987. Proc. Natl. Acad. Sci. USA 84:5730--34 36. EI-Kabbani , 0 . , Chang , C . -H . , Tiede , D . , Norris , J . , Schiffer, M. 1 99 1 . Biochemistry 30:5361-69 37. Marcus, R . A . , Sutin, N. 1985. Biochim. Biophys. Acta 8 1 1 :265-322 3 8 . Allen, J. P . , Feher, G . , Yeates, T. 0 . , Komiya, H . , Rees, D. C. 1988. Proc. Natl. Acad. Sci. USA 85: 848791 39. Swallow, A . J . 1982. See Ref. 106, pp. 59-72 40. Wraight, C. A. 1982. See Ref. 106, pp. 1 8 1-98 4 1 . Prince, R. C . , Dutton, P. L. 1 978. See Ref. 108, pp. 439-53 42. Dutton, P. L . , Leigh, J. S . , Wraight , C . A . 1973. FEBS Lett. 36: 169-73 43. Maroti, P . , Wraight, C. A. 1988. Biochim. Biophys. Acta 934:329--47 44. Rutherford, A. W. , Evans, M. C. w. 1 980. FEBS Lett. 1 1 0:257-61 45. Vermeglio, A. 1 977. Biochim. Biophys . Acta 459:5 1 6-24 46. Wraight , C. A. 1977. Biochim. Biophys. Acta 459:525-3 1 47. Vermeglio, A . , Clayton , R. K. 1977. Biochim. Biophys. Acta 46 1 : 1 59-65 48. Kleinfeld, D. , Okamura, M. Y . , Feher, G . 1984. Biochim. Biophys. Acta 766: 1 26-40 49. Kleinfeld, D . , Okamura, M . Y . , Feher, G. 1985 . Biochim. Biophys. Acta 809: 291-3 1 0 5 0 . McPherson, P. H . , Okamura, M . Y . , Feher, G . 1990. Biochim. Biophys. Acta 1 0 1 6:289-92 5 1 . Clayton, R. K . , Straley , S. C. 1972. Biophys. J. 12: 1 22 1-34 5 2 . Siooten, L. 1 972. Biochim. Biophys . Acta 275:208-1 8 5 3 . Maroti, P . , Wraight, C . A . 1 990. See Ref. 107, pp. 1 . 1 65-68 54. Butler, W. F. , Calvo, R . , Fredkin, D. R . , Isaacson, R . A . , Okamura, M . Y . , Feher, G . 1 984. Biophys. J. 45:94773

895

55. Loach, P. A., Hall, R. L . 1972. Proc. Natl. Acad. Sci. USA 69:786-90 56. Feher, G . , Okamura, M. Y . , McElroy , J. D. 1972. Biochim . Biophys. Acta 267:222-26 57. Hales, B . J . , Case, E. E. 1 98 1 . Biochim. Biophys. Acta 637:291-302 57a. Kleinfeld, D. 1 984. On the dynamics of electron transfer in photosynthetic reac tion centers . PhD thesis. Univ. Calif. , San Diego. 3 1 7 pp. 58. Lubitz , W . , Abresch, E. C . , Debus, R . J . , Isaacson, R. A . , Okamura, M . Y . , Feher, G. 1985 . Biochim. Biophys. Acta 808:464-69 59. Feher, G . , Isaacson, R. A . , Okamura , M. Y . , Lubitz, W. 1985 . In Antennas and Reaction Centers of Photosynthetic Bacteria, ed. M . E. Michel-Beyerle, pp. 1 74-89. Berlin: Springer-Verlag 60. Bagley, K. A . , Abresch, E . , Okamura , M. Y . , Feher, G . , Bauscher, M . , et al. 1990. See Ref. 107, pp. 1. 77-80 6 1 . Buchanan, S . , Michel, H . , Gerwert, K. 1 990. See Ref. 107, pp. 1. 69-72 62. Breton , J . , Thibodeau , D. L . , Berth omieu, C . , Miintele, W . , Vermeglio, A . , Nabedryk, E. 199 1 . FEBS Lett. 278:257-60 63. Hienerwadel, R . , Thibodeau, D . , Lenz, F . , Breton, J . , Nabedryk, E. , et al. 1 99 1 . Fifth Int. Conf. Time Resolved Vibrational Spectrosc. Berlin: Springer Verlag . In press 64. Maroti , P . , Wraight , C. A. 1 988. Biochim. Biophys . Acta 934:3 1 4-28 65. McPherson, P. H . , Okamura, M. Y . , Feher, G . 1988. Biochim. Biophys. Acta 934:348-68 66. Beroza, P. , Fredkin, D. R. , Okamura, M. Y . , Feher, G. 1 99 1 . Proc. Natl. Acad. Sci. USA 88: 5804-8 67. Brzezinski, P., Paddock, M. L. , Messinger, A . , Okamura, M. Y . , Feher, O. 1992. Biophys. 1. Manuscript in preparation 68 . Okamura , M. Y . , Isaacson, R. A . , Feh er, O. 1979. Biochem. Biophys. Acta 546:394--4 1 7 6 9 . Debus, R . J . , Feher, G., Okamura , M . Y. 1986. Biochemistry 25:2276-87 70. Kirmaier, C . , Holten, D . , Debus, R . J . , Feher, G . , Okamura, M. Y. 1 986. Proc. Natl. Acad. Sci. USA 83:640711 7 1 . Takahashi, E . , Wraight, C . A. 1 99 1 . Biochemistry 3 1 : 855-65 72. McPherson, P. H . , Rongey, S. H . , Pad dock, M. L . , Feher, G . , Okamura , M. Y. 199 1 . Biophys. J. 59: 142a 73. Sebban, P. 1988. Biochem . Biophys. Acta 936 : 1 24-32 74. Gao, 1 . -L . , Shapes, R. J . , Wraight, C .


896

OKAMURA & FEHER

A. 1 99 1 . Biochirn . Biophys. Acta 1056:259-72 74a. Paddock, M. 1 99 1 . Characterization of mutant photosynthetic reaction centers from Rhodobacter sphaeroides. PhD thesis. Univ. Calif. , San Diego. 1 55 pp. 75. McPherson, P. H . , Schonfeld, M . , Pad dock, M. L . , Feher, G . , Okamura, M. Y. 1990. Biophys. J. 57:404a 76. Paddock, M. L . , Feher, G . , Okamura, M. Y. 1 990. Biophys. J. 57:569a 77. Paddock, M. L . , Juth, A . , Feher, G . , Okamura, M . Y . 1992. Biophys. J . In 6 1 : 153a 78. Rongey, S . H. , Paddock, M. L., Juth, A. L. , McPherson, P. H . , Feher. G. , Okamura, M. Y. 1 99 1 . Biophys. J. 59: 1 42a 79. Takahashi, E., Wraight, C . A . 1990. FEBS Lett. 283: 140-44 80. Tittor, J . , Soell, C . , Oesterhelt, D . , Butt, H-J . , Bamberg, E. 1 989. EMBO J. 8:3477-82 8 1 . Otto, H . , Marti, T . , Holz, M . , Magi, T . , Lindau, M . , et al. 1989. Proc. Natl. Acad. Sci. USA 86:9228-32 82. Wraight, C. A . , Stein, R. R. 1980. FEBS Lett. 1 1 3:73-77 83. Paddock, M. L . , Rongey, S. H . , McPherson, P. H . , Feher, G . , Okamura, M. Y. 1 99 1 . Biophys. J. 59: 142a 84. Edsall, J. T . , Wyman, J. 1958. Biophys ical Chemistry. New York: Academic. 699 pp. 85 . McPherson, P. H . , Nagaraj an, V . , Par son, W. W . , Okamura, M. Y . , Feher, G. 1990. Biochem. Biophys. Acta 1 01 9:9 1-94 86. Schonfeld, M . , Montal, M . , Feher, G. 1 979. Proc. Nail. Acad. Sci. USA 76:6351-55 87. Packham, N . K . , Mueller, P . , Tiede, D. M . , Dutton, P. L. 1980. FEBS Leu. 1 10: 1 01-6 88. Trissl, H. 1983. Proc. Natl. Acad. Sci. USA 80:71 73-77 89. Feher, G . , Okamura, M. Y. 1984. In Advances in Photosynthesis Research, ed. C. Sybesma, 2 : 1 55-64. Dordrech: Martinus Nijhoff

90. Blatt, Y . , Gopher, A . , Montal , M. , Feh er, G. 1983. Biophys. J. 4 1 : 121a 91. Kaminskaya, O. P . , Drachev, L. A . , Konstantinov , A . A . , Semenov, A . Y . , Skulachev, V . P. 1986. FEBS Lett. 202:224-28 92. Brzezinski, P . , Paddock, M. L . , Rongey, S . H . , Okamura, M . Y . , Feher, G. 1 99 1 . Biophys. J. 59:143a 93. Brzezinski, P., Paddock, M . L., Oka mura, M. Y . , Feher, d. 1 99 1 . Biophys. J, 59: 1 43a 94. Scheiner, S. 1 986. Methods Enzymol. 1 27:456-65 95 . Komiya, H . , Yeates, T. 0. , Rees, D. c . . Allen, J. P . , Feher, G. 1988. Proc. Natl. Acad. Sci. USA 85:90 1 2- 1 6 9 6 . Diner, B . A . , Petrouleas, V . , Wendo laski, J. J. 1 99 1 . Physiol. Plant. 8 1 :423-36 97. Taoka, S . , Crofts, A. R. 1990. See Ref. 1 07 , pp. 1.547-50 98. Blubaugh, D. J . , Govindjee. 1988 . Photosynth. Res. 19:85-128 99. Nagle, J. F . , Tristam-Nagle , S. 1983 . J. Membr. BioI. 74: 1-14 100. Schulten, Z . , Shuiten, K. 1986. Methods Enzymol. 1 27:419-38 1 0 1 . Warshel, A. 1 986. Methods Enzymol. 1 27 :578--87 102. Warshel, A . , Naray-Szabo, G. , Suss man, F. , Hwang, J.-K. 1989. Bioche mistry 28:3629-37 103. Warshel, A . , Aqvist, 1. 1 99 1 . Annu. Rev. Biophys. Biophys. Chem. 20:26798 104. Honig, B. H. , Hubbell, W. L . , Flewell ing, R. F. 1986. Annu. Rev. Biophys . Biophys. Chern. 1 5 : 1 63-93 105. Stewart, R. 1985. The Proton: Applica tions to Organic Chemistry, Vol . 46. New York: Academic. 3 1 3 pp. 106. Trumpower, B. L . , ed. 1982. Function of Quinones in Energy Conserving Sys tems. New York: Academic 1 07 . Baitscheffsky, M . , ed. 1990. Current Research in Photosynthesis, Vol. 1 . Dordrecht: Kluwer 108. Clayton, R. K . , Sistrom, W. R . , eds. 1978. The Photosynthetic Bacteria . New York: Plenum. 946 pp.

Primary photochemistry of reaction centers from the photosynthetic purple bacteria.

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

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

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy.

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

High-resolution structures of photosynthetic reaction centers.

ITO hybrid nanostructure.

Structures of bacterial photosynthetic reaction centers.

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

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

An enriched reaction center preparation from green photosynthetic bacteria.

Electron donors and acceptors in photosynthetic reaction centers.

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

Spectroscopic analysis of genetically modified photosynthetic reaction centers.

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

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

The photochemical electron transfer reactions of photosynthetic bacteria and plants.

Tracking of Proton Transfer Reaction in Supercooled RNA Nucleoside.

Proton-transfer reaction dynamics and energetics in calcification and decalcification.

Proton transfer and isotope-induced reaction in aniline cluster ions.

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

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

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions.

Putative hydrogen bond to tyrosine M208 in photosynthetic reaction centers from Rhodobacter capsulatus significantly slows primary charge separation.