Proc. Natd. Acad. Sci. USA
Vol. 89, pp. 8283-8287, September 1992 Biophysics
Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase (blue copper oxidases/pulse radiolysis)
OLE FARVERt AND ISRAEL PECHTt tDepartment of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tInstitute of General Chemistry, The Royal Danish School of Pharmacy, 2100 Copenhagen, Denmark
Communicated by Harry B. Gray, June 1, 1992
ABSTRACT Anaerobic reduction kinetics of the zucchini squash ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) by pulse radiolytically produced COj radical ions were investigated. Changes in the absorption bands of type 1 [Cu(U)] (610 um) and type 3 [Cu(H)] (330 nm) were monitored over a range of reactant concentrations, pH, and temperature. The direct bimolecular reduction of type 1 [Cu(li)] [(1.2 ± 0.2) x 109 M-1-s-1] was followed by its subsequent reoxidation in three distinct phases, all found to be unimolecular processes with the respective specific rates of 201 ± 8, 20 ± 4, and 2.3 ± 0.2 s"' at pH 5.5 and 298 K. While at this pH no direct bimolecular reduction was resolved in the 330-nm band, at pH 7.0 such a direct process was observed [(6.5 ± 1.2) x 108 M-l s-1]. In the same slower time domains where type 1 [(Cu(I)] reoxidation was monitored, reduction of type 3 [Cu(H)] was observed, which was also concentration independent and with identical rate constants and amplitudes commensurate with those of type 1 [Cu(B)] reoxidation. These results show that after electron uptake by type 1 [Cu(BI], its reoxidation takes place by intramolecular
The reduction potentials of T1 and T3 are identical at 298 K (E' = 350 mV; pH 7.0) (5). The recently determined highresolution three-dimensional model of AO provides unique insights into the detailed structure and spatial relationship among the copper binding centers (6, 7). Thus, AO has been shown to exist as a dimer of identical 70-kDa subunits, each containing one T1, one T2, and one T3 site. These subunits are folded into three interacting domains, all of similar (-barreltype structure, distantly related to the small, blue single copper proteins (6, 7). The T1 site, highly conserved among all blue copper proteins, is within one domain. The T2 and T3 sites were found to be proximal, thus creating a trinuclear center at a distance of 1.23 nm between the T3 copper pair and the T1 site. Ligands coordinated to the T2 and T3 copper ions are provided by the two different domains creating these sites at the interdomain region (6, 7). The catalytic cycle of AO is assumed to proceed by a sequential mechanism in which single electrons are taken up from the respective substrates (e.g., ascorbate) by the T1 [Cu(II)] center. The trinuclear (T2/T3) center has been proposed to be the dioxygen reduction site (1, 2, 5-7). Thus, intramolecular electron transfer (e.t.) from T1 to T3 has long been assumed to be an essential step required to provide the four electrons necessary to yield the final dioxygen reduction product-two water molecules (3, 8, 9). An intramolecular e.t. from T1 [Cu(I)] to the oxidized T3 site in both laccase (10) and AO (11) has recently been reported. Earlier pulse radiolysis studies of the blue oxidases have mainly focused on the kinetics of T1 [Cu(II)] reduction (12-16) and contributed to the notion that the T1 site is the electron uptake site of the blue oxidases. In earlier studies of the blue single copper protein azurin, we have shown that an intramolecular e.t. takes place from the disulfide radical ion, produced by pulse radiolysis, at cystine residues 3-26 to the Cu(II) center (17, 18). This long-range e.t. process provides an instructive model system, yet it has no apparent functional significance in azurins. Hence, we studied the characteristics, particularly the activation barriers, of the e.t. process described above that should proceed through an evolutionary selected pathway, between T1 and T3 in the blue oxidases. We have examined the intramolecular e.t. process from T1 [Cu(I)] to T3 [Cu(II)] in AO as a function of both enzyme reduction state and temperature. We have resolved and separately studied both the intramolecular oxidation of the T1 [Cu(I)] and the concomitant reduction of the T3 [Cu(II)] site. From the temperature dependence of the processes described above, rather low activation enthalpies were calculated.
electron ansfer to type 3 [(Cu(H)J. The observed specific rates
are similar to values reported for the limiting-rate constants of AO reduction by excess substrate, suggesting that internal electron transfer is the rate-determining step ofAO activity. The temperature dependence of the intramolecular electron transfer rate constants was measured from 275 to 308 K at pH 5.5 and, from the Eyring plots, low activation enthalpies were calculat-namely, 9.1 ± 1.1 and 6.8 ± 1.0kJ mol' for the fastest and slowest phases, respectively. The activation entropies observed for these respective phases were -170 ± 9 and -215 ± 16 J-K'1-mol'1. The exceptionally low enthalpy barriers imply the involvement of highiy optimized electron transfer pathways for internal electron transfer.
The blue copper oxidases are widespread redox enzymes that catalyze specific one-electron substrate oxidation by dioxygen, which in turn is reduced to two water molecules (1-3). Their minimal catalytic unit contains four copper ions, which are bound to distinct sites classified according to unique spectroscopic properties (3). The type 1 (Ti) site confers on the bound Cu(II) ions an intense absorption band in the visible region and a rather narrow hyperfine g1l value in the electron paramagnetic resonance (EPR) spectrum. The pair of Cu(II) ions bound to the type 3 (T3) site is characterized by a strong absorbance in the near UV region and by the absence of an EPR signal. The type 2 (T2) Cu(II) site has a rather weak optical absorption spectrum and exhibits a typical hyperfine EPR signal. Ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) was found to be a 140-kDa protein containing eight copper ions per molecule, which can be classified into the above T1, T2, and T3 by their spectroscopic properties (4).
MATERIALS AND METHODS AO isolated from zucchini squash (Cucurbita pepo medullosa) was the product of Boehringer Mannheim. Typically, 100 mg of the enzyme was dissolved in 5-10 ml of 0.01 M
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: AO, ascorbate oxidase; T1, T2, and T3, types 1, 2, and 3 copper binding sites; e.t., electron transfer.
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sodium phosphate buffer (pH 6.0) and passed through a Sephadex G-100 column to remove the stabilizing carbohydrate from the protein. The blue solution obtained was then loaded on a small 12-cm Sephadex C-50 cation-exchange column and washed with the same buffer. The washing was continued with 0.05 M sodium phosphate buffer (still pH 6.0) and, finally, the enzyme was eluted with 0.1 M sodium phosphate (pH 7.0). At this stage, the spectroscopic and enzymatic properties were in accordance with the published literature values (namely, OD610/330 = 0.7; specific activity, -3500 Dawson units per mg of enzyme) (4, 5) and no further purification was deemed necessary. The concentrated enzyme stock solution was used for dilution into appropriate reaction solutions, which typically contained 10 mM sodium phosphate and 0.1 M sodium formate and the enzyme in the range from 5.0 to 14.0 ,uM. Pulse radiolysis experiments were carried out with the Varian V-7715 linear accelerator of the Hebrew University in Jerusalem. Electrons accelerated to 5 MeV were used with pulse lengths in the range from 0.2 to 1.2 As, equivalent to 1-6 ILM COj radical ions. The experimental facility has been described in detail (17, 18). All experiments were carried out under pseudo-first-order conditions with 10-fold or more excess of enzyme sites over the reducing radical concentration. Since the rates of the individual e.t. processes were found to be separated by an order of magnitude, the kinetic analysis was performed by a standard least mean squares method. Analyses ofrates and amplitudes were performed on the Micro PDP1123 computer attached to the accelerator.
Proc. Nad. Acad Sci. USA 89 (1992)
A 0.00 0
'aA 0130.05
0.10 0.5
50
Time, ms
B 0.00 W-
E
0.02
0
.4
0.04 0.06
100 Time, ms
1.0 Time, s
C 0.00
RESULTS Anaerobic solutions of fully oxidized AO (in 0.1 M sodium formate/0.01 mM sodium phosphate, pH 5.5-7.0, saturated with N20) were subjected to sequential pulses of accelerated electrons. The fastest resolvable step after production of the reducing radicals was a bimolecular process of direct e.t. from COj radicals to T1 [Cu(II)]. Fig. 1A illustrates this process as monitored at 610 nm. The pseudo-first-order rate constants derived from these experiments were found to depend on protein and radical concentration yielding a second-order rate constant of (1.2 + 0.2) x 109 M-1-s-1 (298 K; pH 7.0). Approximately one-third of the radical ions reacted with T1 [Cu(II)] of AO, while the rest decayed probably in a competing dimerization reaction to produce oxalate. Thus, a 10 ,uM AO solution could be reduced stepwise by 40-50 pulses until complete reduction of the T1 site was attained. After bimolecular reduction of T1 [Cu(II)J and depending on the extent of AO reduction, either two or three slower reoxidation steps (the fast and slow being of approximately equal amplitudes) were observed by monitoring the 610-nm absorbance (Fig. 1 B-D). From the amplitudes of these absorption changes, it is clear that the T1 [Cu(I)] is only partly reoxidized. The extent of reoxidation depended on the redox state of the AO molecules at the time of the pulse (Fig. 2). Reactivity of the T3 site by CO j radicals was monitored by following the absorbance changes at 330 nm. While no direct reduction of the T3 [Cu(II)] site by the radicals could be resolved at pH 5.5, at pH 7.0 a direct reduction of T3 [Cu(II)] was observed with a pseudo-first-order rate depending on both protein and radical concentrations (Fig. 3). At 285 K, the second-order rate constant is (6.5 + 1.2) x 108 M- Ls-, and its amplitude amounted to less than half of the eventual T3 [Cu(II)] reduction. In the slower time domain, reduction of the 330-nm absorption band was found to proceed in two or three phases at both pH 5.5 and pH 7.0. These took place concomitant with those observed for reoxidation of T1 [Cu(I)] and suggested that reduction of T3 [Cu(II)] is due to oxidation of T1 [Cu(I)]. Indeed, the respective rate constants of these steps were found, within experimental error, to be
B ,
S
0.04
0
4 0.08
0.12 20
2.0
Time, ms
Time, s
D 0.00
am o 0.05 4
0.10
10
0.5
Time, ms
Time,
s
FIG. 1. (A) Time-resolved absorption changes at 610 nm of a 5.3 pM AO (ninth pulse, 28%o reduced T1) solution following pulse radiolysis in N20 saturated 0.01 mM sodium phosphate/0.1 M sodium formate, pH 7.0. Pulse width, 0.8 us; T = 286 K. (B) Time-resolved absorption changes of a 5.5 pM AO solution following the second pulse (5% reduced T1) under the same conditions as in A, except at pH 5.5. Pulse width, 0.5 us; T = 286 K. (C) Time-resolved absorption changes of a 14.0 .M AO solution following the second pulse (4% T1 reduced) under the same conditions as in A, except at pH 5.4. Pulse width, 1.0 ps; T = 276 K. (D) Time-resolved absorption changes of a 5.3 pM AO solution following the thid pulse (7% reduced T1) under the same conditions as in A, except at pH 7.0. Pulse width, 0.8 ps; T = 286 K.
the same. Furthermore, throughout the concentration range studied (2-28 juM T1 [Cu(II)]), the rate constants of both T1
Biophysics: Farver and Pecht
Proc. Nadl. Acad. Sci. USA 89 (1992)
A
1.0
0)
Io
8285
0.00
0
0.5
0.02 0.04
20 Time, ms
0.5
1.0 Time, s
B
1.0
FTIIlolIlI FIG. 2. Extent of reoxidation of the T1 site, calculated as the ratio between the amount of T1 [Cu(I)] being reoxidized and the initial amount of T1 [Cu(II)] reduced following each sequential pulse. Experiments were performed at pH 5.5 and 286 K.
reoxidation and T3 reduction were independent of protein and C02 concentrations (Fig. 4). The amplitudes of the distinct phases vary with the extent of protein reduction (Fig. 2). However, the relative amplitudes of the absorption changes at 610 and 330 nm were constant (AA330/A410 0.65) for these intramolecular reoxidation and reduction phases. Taken together, all these observations clearly show that an intramolecular e.t. process from T1 to T3 is induced after the initial reduction of T1 [Cu(II)]. At pH 5.5 and 298 K, the rate constants observed for the three unimolecular e.t. steps were 201 + 8, 20 + 4, and 2.3 + 0.2 s-1, respectively. No oxidation of T3 [Cu(I)] could be observed during any of the monitored time frames. This confirms the strict anaerobic conditions under which these experiments were performed. The specific rates of the different intramolecular e.t. steps were measured in the pH range 5.5-7.0. Only a 20%o decrease in the respective rate constants with increasing pH was observed. This is in line with the kinetic studies of AO activity by Nakamura et al. (19), who found that enzymatic activity was practically constant between pH 5.5 and 7.0, yet it decreased outside that range. The specific rates of the intramolecular e.t. steps remained concentration independent over the pH range examined, and their temperature dependence was determined at pH 5.5. The Eyring plots derived from the results are presented in Fig. 5. The rather limited temperature dependence yielded activation enthalpies of 9.1 ± 1.1 and 6.8 ± 1.0 kJ-mol-1 for the fast and slow intramolecular e.t. steps, respectively, while the corresponding activation entropies were -170 ± 9 J K-' mol' and -215 ± 16 J K-1 mol-1. The relatively small amplitudes of the intermediate e.t. process precluded satisfactory analysis. Still, no marked temperature dependence of the corresponding rate constant was noticed. Fluoride ions are known to bind specifically to T2 [Cu(II)] of blue oxidases (1, 3, 20, 21) and to inhibit the catalytic activity of these enzymes. We examined the reactivity of 5.5 ,uM AO with CO- radicals after several hours of incubation with a 500-fold excess of F- ions at pH 7.0. A 10% increase in the rate constants of the intramolecular e.t. processes was noticed, which is at the limit of experimental accuracy.
DISCUSSION The identity of the specific rates observed for both reoxidation of T1 [Cu(I)] and reduction of T3 [Cu(II)] combined with their concentration independence and the similarity of their amplitudes clearly support the assignment of the observed processes to intramolecular e.t. from T1 to T3. Meyer et al.
0.00 'r
0.02
0 < 0.04
0.06
20
1.0
Time, ms
Time, s
10
0.5 Time, s
C 0.00
4 0 0.01
0.02
0.03
Time, ms
FIG. 3. (A) Time-resolved absorption changes at 330 nm of a 5.5 ,uM AO solution following pulse radiolysis (fourth pulse, 12% reduced T1) in N20 saturated 0.01 mM phosphate/0.1 M formate, pH 5.5. Pulse width, 0.8 ,us; T = 286 K. (B) Time-resolved absorption changes of a 14.0 ,uM AO solution following the third pulse (7% reduced T1) under the same conditions as in A, except at pH 5.5. Pulse width, 1.0 us; T = 276 K. (C) Time-resolved absorption changes of a 5.3 jLM AO solution following the 27th pulse (90%6 reduced T1) under the same conditions as in A, except at pH 7.0. Pulse width, 0.6 ,us; T = 286 K.
(11) have recently investigated the reduction of oxidized AO by the photochemically produced lumiflavin semiquinone. They observed a second-order direct reduction of the T1 [Cu(II)] by the semiquinone (2.7 x 107 M-1's-1) (pH 7.0) followed by partial reoxidation of the T1 [Cu(I)] site (160 s-1) (pH 7.0; 298 K). Their experimental conditions did not enable monitoring spectral changes at 330 nm, where the presumed electron-accepting T3 [Cu(II)] site absorbs. Still, the latter process, monitored at 610 nm, was interpreted as an intramolecular T1 [Cu(I)] to T3 [Cu(II)] e.t. No further absorption changes at slower time domains were reported (11). Similarly, using pulse radiolytically produced reducing radicals, we observed (10) an intramolecular e.t. from T1 [Cu(I)] to T3 [Cu(II)] in the related blue oxidase Rhus laccase with a rate constant of -1.0 s-1 (pH 7.0; 298 K). The fast-phase intramolecular rate constant is comparable to the reported limiting-rate constants observed for AO
Proc. Nad. Acad. Sci. USA 89 (1992)
Biophysics: Farver and Pecht
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Ishortest e.t. pathway from T1 [Cu(I)J to T3 [Cu(ll)] may be via 1_ ' ' B I ; ~ residues Cys-509 and either His-508 or His-510 (Fig. 6). Both consist of nine covalent bonds, yielding a total 3- pathways 1.34 1 I 180 nm. An alternative pathway is provided by the length of 170 carbonyl oxygen of Cys-509, which is hydrogen bonded to the 160 Ca N8 ofHis-508. Using the method of Beratan et al. (25), we have 7 calculated the relative electronic coupling between the elecC tron donor and acceptor in these two pathways. For e.t. 30 0 20 either of the former, covalent (through bond only) through 10 0 C. pathways, an electronic coupling value of 0.010 was obtained. For the alternative hydrogen-bond-containing pathway, the (U coupling factor becomes 0.014, suggesting that all three path4 3 ways are practically of equal probability. _______if _______i____ 2 From the temperature dependence measurements, we deI B 7 __ 1 t termine AH* = 9.1 kJ-mol-1 and AS" = -170 J K-1-mol-1 for 0 the fastest intramolecular e.t. step. For the slowest one, we 25 30 20 15 10 0 obtained AH* = 6.8 kJ mol-1 and AS" = -215 J K-'.moPQ1. The redox potentials of the T1 and T3 sites were reported to T 1 [Cu(OII) pM be identical at 298 K and pH 7.0 (5). The reaction driving force is small also at pH 5.5 (cf., last paragraph), and we may FIG. 4. First-order rate constants observed forthe the individual calculate the reorganization energy A from the Marcus equal steps of T1 [Cu(I)] reoxi tdation (on syb inls)mediate (o l),and 220 210 200 190
I
reduction (solid symbols) h slow (n. M time scale. restn ictivelv. nlotted as a function of T1 rcE(mi concentration. Experiments were performed at pH 5.5 and 286 K.
reduction at high substrate concentration. Thus, a rate of 120 s l has been reported (22) at pH 7.0 and 298 K with reductic acid as substrate, while with ascorbate at 283 K it was found to be 80-100 s-1 (23). These results are in line with the notion that the rate-limiting step in enzymatic activity is the intramolecular T1 [Cu(I)] to T3 [Cu(II)] e.t. The three-dimensional structure ofAO resolved the thiolate of Cys-509 as one ofthe T1 copper ligands, while the imidazole side chains of the two neighboring amino acids in this polypeptide (His-508 and His-S10) are ligands of the T3 copper ions (6, 7). Thus, it has been proposed (R. Huber and A. Messerschmidt, personal communication; see also ref. 24) that the 1.0 -A 0.8
T
10.6 TO.4
02-
I~~
T
0.2 0.0 3.2
3.4
3.6
3.8
103/T 6.0
tion (26):
Ink
=
Inko
A ..
4RT
1+
AGo -
A
2 -
.8(r ro),
where
kBT ko h
(3is the electronic decay factor and (r - ro) is the edge-to-edge
distance for e.t. Using an edge-to-edge distance between St (Cys-509) and N8 (His-508/510) of 0.90 nm (6) and (3 = 12 nm-1 (26) together with k = 200 s-l for the fast intramolecular e.t. phase, we calculate the reorganization energy A = 132 kJ'molh1. Alternatively, using the through-bond distance (1.34 nm; Fig. 6) and the previously calculated value of( (6.5 nm'1) for long-range e.t. in saturated (-CH2-). chains (27), we find A = 153 kJ mol-1. The above A values are larger than those observed for intramolecular e.t. in other proteins. Thus, for example, the intramolecular e.t. from the disulfide bridge to the T1 [Cu(H)] site in Pseudomonas aeruginosa azurin, a A = 120 k.Fmol-1, has been calculated (17, 18). These larger A values could reflect structural changes of the T3 site occurring upon reduction. The observation of multiple phases in the intramolecular e.t. process is noteworthy and merits further investigation. One possible rationale for this observation could be that it reflects distinct reduction states of the AO molecules. However, no marked difference was observed in the specific rates of the distinct phases as a function of the extent of AO reduction. Therefore, one probably cannot assign these dif-
-B
5.5 T3 Cu 0
5.0
0
OH
4.5
OH
0
0
T3 Cu
4.0r 3.2
3.4
3.6
3.8
o
TI Cu
103/T FIG. 5. (A) Eyring plot of specific rates determined for the fast intramolecular e.t. from Ti [Cu(I)] to T3 [Cu(II)]. (B) Eyring plot of the slow intramolecular e.t. from Ti [Cu(I)] to T3 [Cu(II)].
FIG. 6. Structure of the region separating the T1 and T3 sites in AO according to the atomic model of Messerschmidt et al. (6).
Biophysics: Farver and Pecht ferent steps to the varying reactivity of oxidase molecules reduced to different degrees. Earlier reports, however, have documented the existence of a multiplicity of blue oxidase species with different reactivities. Thus, for example, it is known that in laccase (21) reduction of T2 and T3 [Cu(II)] is inhibited as long as an OH- ion is bound to T2 [Cu(II)]. The dissociation of the OH- is rather slow [a rate constant of 1 s-1 found for laccase and for AO (1, 21), close to the k = 2 s-1 observed for the slowest intramolecular e.t. step in AO]. The high-resolution structure of AO (6, 7) confirmed the presence of water or OH- bound to the T2 and T3 copper sites. However, the hundredfold difference in rate between the fastest and the slowest intramolecular e.t. in AO cannot be due to different reorganization energies at the T3 sites having a coordinated water or OH-, since the activation enthalpies, AH*, of the fastest and slowest intramolecular e.t. phases are, within experimental error, the same. The difference in rate is thus an entropic effect and may be attributed either to the operation of different pathways-i.e., changes that would show up in the product ((r - ro)-or to differences in AS' contributing to the activation entropy term. Indeed, a difference of 45 J-K-1lmol-1 between the activation entropies of the two phases is observed. That no decrease in the rate of internal e.t. is caused by Fions is in line with the notion that the T3 and not T2 site is the primary electron acceptor from the T1 [Cu(I)]. Still, the insensitivity of this process to F- could also be due to the lower affinity of its binding to the T2 site in AO (P. Kroneck, personal communication) and to the presence of excess formate ions. The time course of T1 [Cu(I)] reoxidation fully corresponds with that of T3 [Cu(II)] reduction and raises the long-standing question of the significance of absorption changes monitored at the T3 band: Do the changes at 330 nm correspond to a two-electron reduction of the Cu(II) pair? We have observed (28) that the T3 Cu(II) pair in Rhus laccase can act both as two independent one-electron acceptors as well as a cooperative two-electron acceptor, depending on the reduction potential of the electron donor. Reductive titrations of AO with ascorbate have also indicated equivalence among the single [Cu(II)] ions of T1 and T3 (5). Although the experimental facility used here does not enable the detection of the T2 copper redox state, several lines of evidence exclude the involvement of T2 [Cu(I)] in a concerted two-electron transfer to the T3 site. Each series of pulses was started with a relatively large [protein]/[reductant] ratio. Hence, the probability for a given AO molecule, in the fresh solution, to be reduced by more than a single electron is small (
Vol. 89, pp. 8283-8287, September 1992 Biophysics
Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase (blue copper oxidases/pulse radiolysis)
OLE FARVERt AND ISRAEL PECHTt tDepartment of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tInstitute of General Chemistry, The Royal Danish School of Pharmacy, 2100 Copenhagen, Denmark
Communicated by Harry B. Gray, June 1, 1992
ABSTRACT Anaerobic reduction kinetics of the zucchini squash ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) by pulse radiolytically produced COj radical ions were investigated. Changes in the absorption bands of type 1 [Cu(U)] (610 um) and type 3 [Cu(H)] (330 nm) were monitored over a range of reactant concentrations, pH, and temperature. The direct bimolecular reduction of type 1 [Cu(li)] [(1.2 ± 0.2) x 109 M-1-s-1] was followed by its subsequent reoxidation in three distinct phases, all found to be unimolecular processes with the respective specific rates of 201 ± 8, 20 ± 4, and 2.3 ± 0.2 s"' at pH 5.5 and 298 K. While at this pH no direct bimolecular reduction was resolved in the 330-nm band, at pH 7.0 such a direct process was observed [(6.5 ± 1.2) x 108 M-l s-1]. In the same slower time domains where type 1 [(Cu(I)] reoxidation was monitored, reduction of type 3 [Cu(H)] was observed, which was also concentration independent and with identical rate constants and amplitudes commensurate with those of type 1 [Cu(B)] reoxidation. These results show that after electron uptake by type 1 [Cu(BI], its reoxidation takes place by intramolecular
The reduction potentials of T1 and T3 are identical at 298 K (E' = 350 mV; pH 7.0) (5). The recently determined highresolution three-dimensional model of AO provides unique insights into the detailed structure and spatial relationship among the copper binding centers (6, 7). Thus, AO has been shown to exist as a dimer of identical 70-kDa subunits, each containing one T1, one T2, and one T3 site. These subunits are folded into three interacting domains, all of similar (-barreltype structure, distantly related to the small, blue single copper proteins (6, 7). The T1 site, highly conserved among all blue copper proteins, is within one domain. The T2 and T3 sites were found to be proximal, thus creating a trinuclear center at a distance of 1.23 nm between the T3 copper pair and the T1 site. Ligands coordinated to the T2 and T3 copper ions are provided by the two different domains creating these sites at the interdomain region (6, 7). The catalytic cycle of AO is assumed to proceed by a sequential mechanism in which single electrons are taken up from the respective substrates (e.g., ascorbate) by the T1 [Cu(II)] center. The trinuclear (T2/T3) center has been proposed to be the dioxygen reduction site (1, 2, 5-7). Thus, intramolecular electron transfer (e.t.) from T1 to T3 has long been assumed to be an essential step required to provide the four electrons necessary to yield the final dioxygen reduction product-two water molecules (3, 8, 9). An intramolecular e.t. from T1 [Cu(I)] to the oxidized T3 site in both laccase (10) and AO (11) has recently been reported. Earlier pulse radiolysis studies of the blue oxidases have mainly focused on the kinetics of T1 [Cu(II)] reduction (12-16) and contributed to the notion that the T1 site is the electron uptake site of the blue oxidases. In earlier studies of the blue single copper protein azurin, we have shown that an intramolecular e.t. takes place from the disulfide radical ion, produced by pulse radiolysis, at cystine residues 3-26 to the Cu(II) center (17, 18). This long-range e.t. process provides an instructive model system, yet it has no apparent functional significance in azurins. Hence, we studied the characteristics, particularly the activation barriers, of the e.t. process described above that should proceed through an evolutionary selected pathway, between T1 and T3 in the blue oxidases. We have examined the intramolecular e.t. process from T1 [Cu(I)] to T3 [Cu(II)] in AO as a function of both enzyme reduction state and temperature. We have resolved and separately studied both the intramolecular oxidation of the T1 [Cu(I)] and the concomitant reduction of the T3 [Cu(II)] site. From the temperature dependence of the processes described above, rather low activation enthalpies were calculated.
electron ansfer to type 3 [(Cu(H)J. The observed specific rates
are similar to values reported for the limiting-rate constants of AO reduction by excess substrate, suggesting that internal electron transfer is the rate-determining step ofAO activity. The temperature dependence of the intramolecular electron transfer rate constants was measured from 275 to 308 K at pH 5.5 and, from the Eyring plots, low activation enthalpies were calculat-namely, 9.1 ± 1.1 and 6.8 ± 1.0kJ mol' for the fastest and slowest phases, respectively. The activation entropies observed for these respective phases were -170 ± 9 and -215 ± 16 J-K'1-mol'1. The exceptionally low enthalpy barriers imply the involvement of highiy optimized electron transfer pathways for internal electron transfer.
The blue copper oxidases are widespread redox enzymes that catalyze specific one-electron substrate oxidation by dioxygen, which in turn is reduced to two water molecules (1-3). Their minimal catalytic unit contains four copper ions, which are bound to distinct sites classified according to unique spectroscopic properties (3). The type 1 (Ti) site confers on the bound Cu(II) ions an intense absorption band in the visible region and a rather narrow hyperfine g1l value in the electron paramagnetic resonance (EPR) spectrum. The pair of Cu(II) ions bound to the type 3 (T3) site is characterized by a strong absorbance in the near UV region and by the absence of an EPR signal. The type 2 (T2) Cu(II) site has a rather weak optical absorption spectrum and exhibits a typical hyperfine EPR signal. Ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) was found to be a 140-kDa protein containing eight copper ions per molecule, which can be classified into the above T1, T2, and T3 by their spectroscopic properties (4).
MATERIALS AND METHODS AO isolated from zucchini squash (Cucurbita pepo medullosa) was the product of Boehringer Mannheim. Typically, 100 mg of the enzyme was dissolved in 5-10 ml of 0.01 M
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: AO, ascorbate oxidase; T1, T2, and T3, types 1, 2, and 3 copper binding sites; e.t., electron transfer.
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8284
Biophysics: Farver and Pecht
sodium phosphate buffer (pH 6.0) and passed through a Sephadex G-100 column to remove the stabilizing carbohydrate from the protein. The blue solution obtained was then loaded on a small 12-cm Sephadex C-50 cation-exchange column and washed with the same buffer. The washing was continued with 0.05 M sodium phosphate buffer (still pH 6.0) and, finally, the enzyme was eluted with 0.1 M sodium phosphate (pH 7.0). At this stage, the spectroscopic and enzymatic properties were in accordance with the published literature values (namely, OD610/330 = 0.7; specific activity, -3500 Dawson units per mg of enzyme) (4, 5) and no further purification was deemed necessary. The concentrated enzyme stock solution was used for dilution into appropriate reaction solutions, which typically contained 10 mM sodium phosphate and 0.1 M sodium formate and the enzyme in the range from 5.0 to 14.0 ,uM. Pulse radiolysis experiments were carried out with the Varian V-7715 linear accelerator of the Hebrew University in Jerusalem. Electrons accelerated to 5 MeV were used with pulse lengths in the range from 0.2 to 1.2 As, equivalent to 1-6 ILM COj radical ions. The experimental facility has been described in detail (17, 18). All experiments were carried out under pseudo-first-order conditions with 10-fold or more excess of enzyme sites over the reducing radical concentration. Since the rates of the individual e.t. processes were found to be separated by an order of magnitude, the kinetic analysis was performed by a standard least mean squares method. Analyses ofrates and amplitudes were performed on the Micro PDP1123 computer attached to the accelerator.
Proc. Nad. Acad Sci. USA 89 (1992)
A 0.00 0
'aA 0130.05
0.10 0.5
50
Time, ms
B 0.00 W-
E
0.02
0
.4
0.04 0.06
100 Time, ms
1.0 Time, s
C 0.00
RESULTS Anaerobic solutions of fully oxidized AO (in 0.1 M sodium formate/0.01 mM sodium phosphate, pH 5.5-7.0, saturated with N20) were subjected to sequential pulses of accelerated electrons. The fastest resolvable step after production of the reducing radicals was a bimolecular process of direct e.t. from COj radicals to T1 [Cu(II)]. Fig. 1A illustrates this process as monitored at 610 nm. The pseudo-first-order rate constants derived from these experiments were found to depend on protein and radical concentration yielding a second-order rate constant of (1.2 + 0.2) x 109 M-1-s-1 (298 K; pH 7.0). Approximately one-third of the radical ions reacted with T1 [Cu(II)] of AO, while the rest decayed probably in a competing dimerization reaction to produce oxalate. Thus, a 10 ,uM AO solution could be reduced stepwise by 40-50 pulses until complete reduction of the T1 site was attained. After bimolecular reduction of T1 [Cu(II)J and depending on the extent of AO reduction, either two or three slower reoxidation steps (the fast and slow being of approximately equal amplitudes) were observed by monitoring the 610-nm absorbance (Fig. 1 B-D). From the amplitudes of these absorption changes, it is clear that the T1 [Cu(I)] is only partly reoxidized. The extent of reoxidation depended on the redox state of the AO molecules at the time of the pulse (Fig. 2). Reactivity of the T3 site by CO j radicals was monitored by following the absorbance changes at 330 nm. While no direct reduction of the T3 [Cu(II)] site by the radicals could be resolved at pH 5.5, at pH 7.0 a direct reduction of T3 [Cu(II)] was observed with a pseudo-first-order rate depending on both protein and radical concentrations (Fig. 3). At 285 K, the second-order rate constant is (6.5 + 1.2) x 108 M- Ls-, and its amplitude amounted to less than half of the eventual T3 [Cu(II)] reduction. In the slower time domain, reduction of the 330-nm absorption band was found to proceed in two or three phases at both pH 5.5 and pH 7.0. These took place concomitant with those observed for reoxidation of T1 [Cu(I)] and suggested that reduction of T3 [Cu(II)] is due to oxidation of T1 [Cu(I)]. Indeed, the respective rate constants of these steps were found, within experimental error, to be
B ,
S
0.04
0
4 0.08
0.12 20
2.0
Time, ms
Time, s
D 0.00
am o 0.05 4
0.10
10
0.5
Time, ms
Time,
s
FIG. 1. (A) Time-resolved absorption changes at 610 nm of a 5.3 pM AO (ninth pulse, 28%o reduced T1) solution following pulse radiolysis in N20 saturated 0.01 mM sodium phosphate/0.1 M sodium formate, pH 7.0. Pulse width, 0.8 us; T = 286 K. (B) Time-resolved absorption changes of a 5.5 pM AO solution following the second pulse (5% reduced T1) under the same conditions as in A, except at pH 5.5. Pulse width, 0.5 us; T = 286 K. (C) Time-resolved absorption changes of a 14.0 .M AO solution following the second pulse (4% T1 reduced) under the same conditions as in A, except at pH 5.4. Pulse width, 1.0 ps; T = 276 K. (D) Time-resolved absorption changes of a 5.3 pM AO solution following the thid pulse (7% reduced T1) under the same conditions as in A, except at pH 7.0. Pulse width, 0.8 ps; T = 286 K.
the same. Furthermore, throughout the concentration range studied (2-28 juM T1 [Cu(II)]), the rate constants of both T1
Biophysics: Farver and Pecht
Proc. Nadl. Acad. Sci. USA 89 (1992)
A
1.0
0)
Io
8285
0.00
0
0.5
0.02 0.04
20 Time, ms
0.5
1.0 Time, s
B
1.0
FTIIlolIlI FIG. 2. Extent of reoxidation of the T1 site, calculated as the ratio between the amount of T1 [Cu(I)] being reoxidized and the initial amount of T1 [Cu(II)] reduced following each sequential pulse. Experiments were performed at pH 5.5 and 286 K.
reoxidation and T3 reduction were independent of protein and C02 concentrations (Fig. 4). The amplitudes of the distinct phases vary with the extent of protein reduction (Fig. 2). However, the relative amplitudes of the absorption changes at 610 and 330 nm were constant (AA330/A410 0.65) for these intramolecular reoxidation and reduction phases. Taken together, all these observations clearly show that an intramolecular e.t. process from T1 to T3 is induced after the initial reduction of T1 [Cu(II)]. At pH 5.5 and 298 K, the rate constants observed for the three unimolecular e.t. steps were 201 + 8, 20 + 4, and 2.3 + 0.2 s-1, respectively. No oxidation of T3 [Cu(I)] could be observed during any of the monitored time frames. This confirms the strict anaerobic conditions under which these experiments were performed. The specific rates of the different intramolecular e.t. steps were measured in the pH range 5.5-7.0. Only a 20%o decrease in the respective rate constants with increasing pH was observed. This is in line with the kinetic studies of AO activity by Nakamura et al. (19), who found that enzymatic activity was practically constant between pH 5.5 and 7.0, yet it decreased outside that range. The specific rates of the intramolecular e.t. steps remained concentration independent over the pH range examined, and their temperature dependence was determined at pH 5.5. The Eyring plots derived from the results are presented in Fig. 5. The rather limited temperature dependence yielded activation enthalpies of 9.1 ± 1.1 and 6.8 ± 1.0 kJ-mol-1 for the fast and slow intramolecular e.t. steps, respectively, while the corresponding activation entropies were -170 ± 9 J K-' mol' and -215 ± 16 J K-1 mol-1. The relatively small amplitudes of the intermediate e.t. process precluded satisfactory analysis. Still, no marked temperature dependence of the corresponding rate constant was noticed. Fluoride ions are known to bind specifically to T2 [Cu(II)] of blue oxidases (1, 3, 20, 21) and to inhibit the catalytic activity of these enzymes. We examined the reactivity of 5.5 ,uM AO with CO- radicals after several hours of incubation with a 500-fold excess of F- ions at pH 7.0. A 10% increase in the rate constants of the intramolecular e.t. processes was noticed, which is at the limit of experimental accuracy.
DISCUSSION The identity of the specific rates observed for both reoxidation of T1 [Cu(I)] and reduction of T3 [Cu(II)] combined with their concentration independence and the similarity of their amplitudes clearly support the assignment of the observed processes to intramolecular e.t. from T1 to T3. Meyer et al.
0.00 'r
0.02
0 < 0.04
0.06
20
1.0
Time, ms
Time, s
10
0.5 Time, s
C 0.00
4 0 0.01
0.02
0.03
Time, ms
FIG. 3. (A) Time-resolved absorption changes at 330 nm of a 5.5 ,uM AO solution following pulse radiolysis (fourth pulse, 12% reduced T1) in N20 saturated 0.01 mM phosphate/0.1 M formate, pH 5.5. Pulse width, 0.8 ,us; T = 286 K. (B) Time-resolved absorption changes of a 14.0 ,uM AO solution following the third pulse (7% reduced T1) under the same conditions as in A, except at pH 5.5. Pulse width, 1.0 us; T = 276 K. (C) Time-resolved absorption changes of a 5.3 jLM AO solution following the 27th pulse (90%6 reduced T1) under the same conditions as in A, except at pH 7.0. Pulse width, 0.6 ,us; T = 286 K.
(11) have recently investigated the reduction of oxidized AO by the photochemically produced lumiflavin semiquinone. They observed a second-order direct reduction of the T1 [Cu(II)] by the semiquinone (2.7 x 107 M-1's-1) (pH 7.0) followed by partial reoxidation of the T1 [Cu(I)] site (160 s-1) (pH 7.0; 298 K). Their experimental conditions did not enable monitoring spectral changes at 330 nm, where the presumed electron-accepting T3 [Cu(II)] site absorbs. Still, the latter process, monitored at 610 nm, was interpreted as an intramolecular T1 [Cu(I)] to T3 [Cu(II)] e.t. No further absorption changes at slower time domains were reported (11). Similarly, using pulse radiolytically produced reducing radicals, we observed (10) an intramolecular e.t. from T1 [Cu(I)] to T3 [Cu(II)] in the related blue oxidase Rhus laccase with a rate constant of -1.0 s-1 (pH 7.0; 298 K). The fast-phase intramolecular rate constant is comparable to the reported limiting-rate constants observed for AO
Proc. Nad. Acad. Sci. USA 89 (1992)
Biophysics: Farver and Pecht
8286
Ishortest e.t. pathway from T1 [Cu(I)J to T3 [Cu(ll)] may be via 1_ ' ' B I ; ~ residues Cys-509 and either His-508 or His-510 (Fig. 6). Both consist of nine covalent bonds, yielding a total 3- pathways 1.34 1 I 180 nm. An alternative pathway is provided by the length of 170 carbonyl oxygen of Cys-509, which is hydrogen bonded to the 160 Ca N8 ofHis-508. Using the method of Beratan et al. (25), we have 7 calculated the relative electronic coupling between the elecC tron donor and acceptor in these two pathways. For e.t. 30 0 20 either of the former, covalent (through bond only) through 10 0 C. pathways, an electronic coupling value of 0.010 was obtained. For the alternative hydrogen-bond-containing pathway, the (U coupling factor becomes 0.014, suggesting that all three path4 3 ways are practically of equal probability. _______if _______i____ 2 From the temperature dependence measurements, we deI B 7 __ 1 t termine AH* = 9.1 kJ-mol-1 and AS" = -170 J K-1-mol-1 for 0 the fastest intramolecular e.t. step. For the slowest one, we 25 30 20 15 10 0 obtained AH* = 6.8 kJ mol-1 and AS" = -215 J K-'.moPQ1. The redox potentials of the T1 and T3 sites were reported to T 1 [Cu(OII) pM be identical at 298 K and pH 7.0 (5). The reaction driving force is small also at pH 5.5 (cf., last paragraph), and we may FIG. 4. First-order rate constants observed forthe the individual calculate the reorganization energy A from the Marcus equal steps of T1 [Cu(I)] reoxi tdation (on syb inls)mediate (o l),and 220 210 200 190
I
reduction (solid symbols) h slow (n. M time scale. restn ictivelv. nlotted as a function of T1 rcE(mi concentration. Experiments were performed at pH 5.5 and 286 K.
reduction at high substrate concentration. Thus, a rate of 120 s l has been reported (22) at pH 7.0 and 298 K with reductic acid as substrate, while with ascorbate at 283 K it was found to be 80-100 s-1 (23). These results are in line with the notion that the rate-limiting step in enzymatic activity is the intramolecular T1 [Cu(I)] to T3 [Cu(II)] e.t. The three-dimensional structure ofAO resolved the thiolate of Cys-509 as one ofthe T1 copper ligands, while the imidazole side chains of the two neighboring amino acids in this polypeptide (His-508 and His-S10) are ligands of the T3 copper ions (6, 7). Thus, it has been proposed (R. Huber and A. Messerschmidt, personal communication; see also ref. 24) that the 1.0 -A 0.8
T
10.6 TO.4
02-
I~~
T
0.2 0.0 3.2
3.4
3.6
3.8
103/T 6.0
tion (26):
Ink
=
Inko
A ..
4RT
1+
AGo -
A
2 -
.8(r ro),
where
kBT ko h
(3is the electronic decay factor and (r - ro) is the edge-to-edge
distance for e.t. Using an edge-to-edge distance between St (Cys-509) and N8 (His-508/510) of 0.90 nm (6) and (3 = 12 nm-1 (26) together with k = 200 s-l for the fast intramolecular e.t. phase, we calculate the reorganization energy A = 132 kJ'molh1. Alternatively, using the through-bond distance (1.34 nm; Fig. 6) and the previously calculated value of( (6.5 nm'1) for long-range e.t. in saturated (-CH2-). chains (27), we find A = 153 kJ mol-1. The above A values are larger than those observed for intramolecular e.t. in other proteins. Thus, for example, the intramolecular e.t. from the disulfide bridge to the T1 [Cu(H)] site in Pseudomonas aeruginosa azurin, a A = 120 k.Fmol-1, has been calculated (17, 18). These larger A values could reflect structural changes of the T3 site occurring upon reduction. The observation of multiple phases in the intramolecular e.t. process is noteworthy and merits further investigation. One possible rationale for this observation could be that it reflects distinct reduction states of the AO molecules. However, no marked difference was observed in the specific rates of the distinct phases as a function of the extent of AO reduction. Therefore, one probably cannot assign these dif-
-B
5.5 T3 Cu 0
5.0
0
OH
4.5
OH
0
0
T3 Cu
4.0r 3.2
3.4
3.6
3.8
o
TI Cu
103/T FIG. 5. (A) Eyring plot of specific rates determined for the fast intramolecular e.t. from Ti [Cu(I)] to T3 [Cu(II)]. (B) Eyring plot of the slow intramolecular e.t. from Ti [Cu(I)] to T3 [Cu(II)].
FIG. 6. Structure of the region separating the T1 and T3 sites in AO according to the atomic model of Messerschmidt et al. (6).
Biophysics: Farver and Pecht ferent steps to the varying reactivity of oxidase molecules reduced to different degrees. Earlier reports, however, have documented the existence of a multiplicity of blue oxidase species with different reactivities. Thus, for example, it is known that in laccase (21) reduction of T2 and T3 [Cu(II)] is inhibited as long as an OH- ion is bound to T2 [Cu(II)]. The dissociation of the OH- is rather slow [a rate constant of 1 s-1 found for laccase and for AO (1, 21), close to the k = 2 s-1 observed for the slowest intramolecular e.t. step in AO]. The high-resolution structure of AO (6, 7) confirmed the presence of water or OH- bound to the T2 and T3 copper sites. However, the hundredfold difference in rate between the fastest and the slowest intramolecular e.t. in AO cannot be due to different reorganization energies at the T3 sites having a coordinated water or OH-, since the activation enthalpies, AH*, of the fastest and slowest intramolecular e.t. phases are, within experimental error, the same. The difference in rate is thus an entropic effect and may be attributed either to the operation of different pathways-i.e., changes that would show up in the product ((r - ro)-or to differences in AS' contributing to the activation entropy term. Indeed, a difference of 45 J-K-1lmol-1 between the activation entropies of the two phases is observed. That no decrease in the rate of internal e.t. is caused by Fions is in line with the notion that the T3 and not T2 site is the primary electron acceptor from the T1 [Cu(I)]. Still, the insensitivity of this process to F- could also be due to the lower affinity of its binding to the T2 site in AO (P. Kroneck, personal communication) and to the presence of excess formate ions. The time course of T1 [Cu(I)] reoxidation fully corresponds with that of T3 [Cu(II)] reduction and raises the long-standing question of the significance of absorption changes monitored at the T3 band: Do the changes at 330 nm correspond to a two-electron reduction of the Cu(II) pair? We have observed (28) that the T3 Cu(II) pair in Rhus laccase can act both as two independent one-electron acceptors as well as a cooperative two-electron acceptor, depending on the reduction potential of the electron donor. Reductive titrations of AO with ascorbate have also indicated equivalence among the single [Cu(II)] ions of T1 and T3 (5). Although the experimental facility used here does not enable the detection of the T2 copper redox state, several lines of evidence exclude the involvement of T2 [Cu(I)] in a concerted two-electron transfer to the T3 site. Each series of pulses was started with a relatively large [protein]/[reductant] ratio. Hence, the probability for a given AO molecule, in the fresh solution, to be reduced by more than a single electron is small (
