Electrostatic Interaction between Redox Cofactors in Photosynthetic Reaction Centers
2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês
10.1074/jbc.m408888200
ISSN1083-351X
AutoresJean Alric, Aude Cuni, Hideaki Maki, Kenji V. P. Nagashima, André Verméglio, Fabrice Rappaport,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoIntramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921–3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them. Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921–3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them. Proteins exert a fine electrochemical tuning of the redox potential of the cofactors they bind in order to perform the various electron transfer reactions that are involved in biological processes. As a famous example, the redox potentials (Em) 1The abbreviations used are: Em, midpoint redox potential; Eh, ambient redox potential; P, primary electron donor; RC, reaction center; WT, wild type; MOPS, 4-morpholinepropanesulfonic acid; g2, 2 tensor component.1The abbreviations used are: Em, midpoint redox potential; Eh, ambient redox potential; P, primary electron donor; RC, reaction center; WT, wild type; MOPS, 4-morpholinepropanesulfonic acid; g2, 2 tensor component. of c-type cytochromes are tuned by more than 500 mV (see Ref. 1Gunner M.R. Alexov E. Torres E. Lipovaca S. J. Biol. Inorg. Chem. 1997; 2: 126-134Crossref Scopus (84) Google Scholar and references therein). The physicochemical basis of a such wide range of modulations has been rationalized by various authors who all agree that the protein medium has the unique property of providing a dielectric environment in which the redox cofactors are embedded with an intricate charge or dipole distribution (1Gunner M.R. Alexov E. Torres E. Lipovaca S. J. Biol. Inorg. Chem. 1997; 2: 126-134Crossref Scopus (84) Google Scholar, 2Parson W.W. Chu Z.-T. Warshel A. Biochim. Biophys. Acta. 1990; 1017: 251-272Crossref PubMed Scopus (239) Google Scholar, 3Warshel A. Aqvist J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 267-298Crossref PubMed Scopus (415) Google Scholar). From a general standpoint, the free energy difference between the oxidized and reduced forms of any redox cofactor in a protein is the sum of several terms. Among these are ΔGconf and ΔGel. ΔGconf accounts for any conformational change that the change in the redox state of the cofactor may induce (including proton or ion binding or release). ΔGel results from the electrostatic potential at the cofactors resulting from the individual charged groups and the permanent dipoles within the protein. Estimating the respective values of these different terms is a difficult task, yet their sum is readily accessible experimentally because it can be obtained by comparing the absolute values of the Em in solution and in the protein. The latter may be obtained by two different methods. The most commonly used one is equilibrium redox titration. The other relies on the determination, by a functional analysis, of the free energy change associated with an electron transfer reaction between two cofactors. Such a change in free energy is generally considered as equal to the difference in Ems between the electron donor and acceptor. Thus, provided that one of these two redox potentials is known, the other one is readily inferred. However, these two methods sometimes yield different results and this has led to the distinction between "equilibrium redox potential" and "operating redox potential." These differences arise because the two methods do not probe the same state of the redox cofactors. Two types of phenomena may account for these differences. One comes from the distinct time domain involved in equilibrium redox titration and functional analysis. Whereas redox titrations require thermodynamic equilibrium between the sample and the solution poised at a given potential, the functional analysis allows one to probe transient states whose free energy may differ significantly from that of the equilibrated states. Indeed, in response to the change in the redox state of a given cofactor, the protein environment may undergo energetic relaxation (such as proton transfer, conformational changes), which may be slower than the lifetime of the transient oxidized (or reduced) cofactor. Armstrong et al. (4Armstrong F.A. Camba R. Heering H.A. Hirst J. Jeuken L.J. Jones A.K. Leger C. McEvoy J.P. Faraday Discuss. Chem. Soc. 2000; 116: 191-203Crossref Scopus (87) Google Scholar) have nicely illustrated this point with the "fast-scan electrovoltammetric" technique. Other examples are found in photosynthetic reaction centers (RC) (for example, see Refs. 5Woodbury N.W. Parson W.W. Gunner M.R. Prince R.C. Dutton P.L. Biochim. Biophys. Acta. 1986; 851: 6-22Crossref PubMed Scopus (185) Google Scholar and 6Sebban P. Wraight C.A. Biochim. Biophys. Acta. 1989; 974: 54-65Crossref Scopus (61) Google Scholar). An alternative but non-exclusive explanation of the different Ems yielded by redox titration and functional analysis relies on the fact that most of the membrane proteins involved in electron transfer reactions bind several cofactors, which are usually located at less than 15 Å, one from the other. Such short distances may result in significant electrostatic interactions among the different cofactors. Thus, for a given cofactor, ΔGel includes the electrostatic contributions of the nearby electron carriers. Such a contribution has been nicely illustrated in the case of the tetraheme cytochrome of Blastochloris (formerly Rhodopseudomonas) viridis (7Gunner M.R. Honig B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9151-9155Crossref PubMed Scopus (174) Google Scholar, 8Nitschke W. Rutherford A.W. Biochem. Soc. Trans. 1994; 22: 694-699Crossref PubMed Scopus (9) Google Scholar). However, it is noteworthy that throughout a redox titration, all of the cofactors undergo an identical charge change in terms of sign (i.e. all are either reduced or oxidized), whereas in an electron transfer chain, two nearby cofactors involved in an electron transfer reaction will undergo charge changes of opposite sign (one will be oxidized at the expense of the other). Consequently, if the electrostatic interaction between the two is significant, the difference between their equilibrium Ems will be greater than the free energy change associated with the electron transfer between them. In this paper, we illustrated the importance of such electrostatic interactions in electron transfer chains. Although electron transfer chains embedded in a single protein are found in many biological pathways, the photosynthetic chains are ideally suited for such studies. Indeed, not only do they allow redox titration of the various redox cofactors, but also the kinetics of electron transfer reaction may be characterized with an unequalled time resolution. In particular, the bacterial photosynthetic RC of B. viridis and its associated tetraheme cytochrome have been intensively studied. Its three-dimensional structure has been solved (9Deisenhofer J. Epp O. Miki K. Huber R. Michel H. Nature. 1985; 318: 618-624Crossref PubMed Scopus (2560) Google Scholar), and the spectroscopic or redox properties of the various cofactors are known (see Ref. 10Nitschke W. Dracheva S.M. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 775-805Google Scholar for a review). Further, the different electron transfer steps have been characterized (11Ortega J.M. Mathis P. Biochemistry. 1993; 32: 1141-1151Crossref PubMed Scopus (76) Google Scholar). In membrane fragments as well as purified RC of B. viridis, the reduction of the oxidized primary electron donor P+ by the closest heme, c559, is multiphasic (12Ortega J.M. Mathis P. Photosynth. Res. 1992; 34: 127Google Scholar). Interestingly, this feature has been interpreted along the lines of either of the two phenomena that has just been described, a conformational heterogeneity yielding a distribution of substates (11Ortega J.M. Mathis P. Biochemistry. 1993; 32: 1141-1151Crossref PubMed Scopus (76) Google Scholar) or a low equilibrium constant of this electron transfer reaction (13Baymann F. Rappaport F. Biochemistry. 1998; 37: 15320-15326Crossref PubMed Scopus (10) Google Scholar). To reconcile this latter hypothesis with the equilibrium constant of ∼100 expected from the difference in midpoint potentials of the P+/P and c559+/c559, Baymann and Rappaport (13Baymann F. Rappaport F. Biochemistry. 1998; 37: 15320-15326Crossref PubMed Scopus (10) Google Scholar) proposed that an electrostatic interaction between P and its closest heme raises the redox potential of the P+/P couple. As discussed below, the present results support this hypothesis. From an experimental standpoint, B. viridis has the drawback of being unable to grow heterotrophically, making the mutagenesis approach uncertain despite a few successful attempts. Recently, Maki et al. (14Maki H. Matsuura K. Shimada K. Nagashima K.V.P. J. Biol. Chem. 2003; 278: 3921-3928Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) succeeded in transferring the membrane bound cytochrome of B. viridis to another bacterium, Rubrivivax gelatinosus, in which mutagenesis strategy may be planned (15Nagashima K.V.P. Shimada K. Matsuura K. FEBS Lett. 1996; 385: 209-213Crossref PubMed Scopus (26) Google Scholar, 16Nagashima K.V.P. Matsuura K. Shimada K. Verméglio A. Biochemistry. 2002; 41: 14028-14032Crossref PubMed Scopus (16) Google Scholar). In this paper, we further investigated this chimeric RC (hereafter named VC-F) and show that most of the various cofactors at the donor side of this RC show absorption spectra and relative orientations similar to those found in the respective native RCs, indicating that the backbone structure of the protein as well as the interactions between the hemes and the side chains are conserved (Fig. 1). These findings make the chimeric RC suited for the direct characterization of electrostatic interactions between cofactors by the mean of equilibrium redox titration. Indeed, in this RC, two cofactors (the primary electron donor (P) and the closest heme c559) are expected to have redox potentials differing by less than 50 mV (see Fig. 1). Such a case allows the direct observation of a putative electrostatic interaction, because it should manifest itself by a deviation to a one-electron Nernst curve. This contrasts with the case where the two interacting redox centers have strongly different midpoint potentials (as in the B. viridis case, for example) for they are then expected to titrate in well separated redox potential ranges and thus yield titration curves that follow the classical Nernst equation. Accordingly, we found that the redox titration of the primary donor (P/P+) of the chimeric RC displayed a deviation to a one-electron Nernst curve. The redox potential of the primary donor was downshifted by 50 mV in the chimera with respect to the WT R. gelatinosus strain. Interestingly, the redox potential of the closest heme (the c559 heme) underwent a converse upshift of similar amplitude. These data prove that electrostatic interactions between cofactors in proteins modulate their redox potentials. Interspecific replacement of the gene coding for the RC-bound cytochrome subunit in R. gelatinosus used to produce VC-F mutant is described by Maki et al. (14Maki H. Matsuura K. Shimada K. Nagashima K.V.P. J. Biol. Chem. 2003; 278: 3921-3928Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The site-directed substitution by a two-step PCR method of Arg-204 for a Leu in the cytochrome subunit of the VC-F strain is described by Nagashima et al. (17Nagashima K.V.P. Alric J. Matsuura K. Shimada K. Verméglio A. Bruce D. van der Est A. 13th International Congress on Photosynthesis. Allen Press, Montréal2004Google Scholar). Cells of the VC-F mutant of R. gelatinosus were grown for 24 h in the light and in anaerobic conditions in Hutner medium with 50 μg·ml–1 kanamycin and 20 μg·ml–1 ampicillin. For membrane preparations, cells were harvested by centrifugation at 4000 × g for 10 min, resuspended in 20 mm Tris-HCl (pH 7), and disrupted by a French press at 50 megapascals. The remaining intact cells were separated from the membrane supernatant by centrifugation at 10,000 × g for 10 min. The membrane fragments then were collected after centrifugation at 250,000 × g for 90 min and resuspended in 20 mm Tris-HCl, 100 mm KCl (pH 7), for equilibrium redox titrations. EPR Spectroscopy—Membrane fragments were resuspended in 20 mm MOPS (pH 7) and oxidized by the addition of 2 mm potassium ferricyanide. The membranes were then washed free from ferricyanide by renewed pelleting and resuspension in 20 mm MOPS (pH 7). Electron paramagnetic response spectra were taken at 15 K using a Bruker ER 300 X-band spectrometer equipped with an Oxford Instruments helium cryostat and temperature-control system. The instrument settings were as follows: microwave power, 6.7 milliwatts; microwave frequency, 9.43 GHz; and modulation amplitude, 2.5 millitesla. Angular dependence of EPR signals was investigated on oriented membrane multilayer obtained by drying the membrane fragments onto Mylar sheets (18Rutherford A.W. Sétif P. Biochim. Biophys. Acta. 1990; 1019: 128-132Crossref Scopus (51) Google Scholar). Redox titrations were performed as described previously (13Baymann F. Rappaport F. Biochemistry. 1998; 37: 15320-15326Crossref PubMed Scopus (10) Google Scholar, 19Baymann F. Moss D.A. Mantele W. Anal. Biochem. 1991; 199: 269-274Crossref PubMed Scopus (50) Google Scholar) in an electrochemical cell (100-μm optical path length) with three electrodes: a platinum electrode; a gold grid (InterNet Inc.) modified by PATS (2-pyridinecarboxaldehyde thiosemicarbazone, Sigma) to avoid irreversible adsorption of the proteins onto the gold grid; and an Ag/AgCl reference electrode in 3 m KCl. The redox mediators were used at 20 μm each: 1,4-benzoquinone (Em = +280 mV); 1,1-dimethyl ferrocene (Em = +340 mV); ferrocene (Em = +420 mV); and monocarboxylic acid ferrocene (Em = +530 mV). The optical spectroscopic measurements were performed on two different laboratory-built absorption spectrophotometers: a Xenon flashlamp microsecond time resolution one (20Joliot P. Béal D. Frilley B. J. Chim. Phys. 1980; 77: 209-216Crossref Google Scholar) used for the titration of the cytochromes in the α-band and a Nd:YAG-pulsed laser nanosecond time resolution one (21Béal D. Rappaport F. Joliot P. Rev. Sci. Instrum. 1999; 70: 202-207Crossref Scopus (80) Google Scholar) used for the kinetic experiments and the flash-induced titration of the primary electron donor. Redox Characteristics of the P+/P Couple in the WT and Chimeric RCs—The primary electron donor P was titrated in membrane fragments purified from R. gelatinosus and B. viridis WT strains and the chimeric VC-F strain (Fig. 2, left panel) by measuring the flash-induced absorption changes at 605 nm, 50 ns after the actinic flash. Both the oxidative or reducing waves yielded similar results, indicating that thermodynamic equilibrium was reached during the titration (data not shown). The titration curve in B. viridis could be satisfyingly fitted with a Nernst curve with Em = +500 mV. The P+/P couple in R. gelatinosus titrates with an Em of ∼+400 mV, consistent with previous findings (22Dutton P.L. Biochim. Biophys. Acta. 1971; 226: 63-80Crossref PubMed Scopus (347) Google Scholar), but, interestingly, the titration curve showed a slight deviation to a one-electron Nernst equation (dotted line). In the VC-F-chimeric RC, the results were significantly different from that in R. gelatinosus WT. (i) The deviation to the one-electron Nernst equation (dotted line) was more pronounced. (ii) The overall midpoint potential was lower by ∼50 mV. Several non-exclusive hypotheses may account for such a difference. The first one relies on a strong heterogeneity among the RCs. As a possible reason for such heterogeneity, one may consider that the tetraheme subunit is lost in a fraction of RCs during the preparation of the membrane fragments. To test this hypothesis, we measured (Fig. 3) the amount of long-lived P+ under conditions where only the c556 and c559 hemes were reduced in the dark (Fig. 3, squares) and under conditions where the high and low potential hemes were reduced in the dark (Fig. 3, triangles). Whereas in the former case ∼50% P+ was still present 200 μs after the flash, this fraction of long-lived P+ was only 3% when the low potential hemes were reduced. Under these latter conditions, the free energy change associated with P+ reduction is expected to be large because of the low midpoint potentials of the electron donor (see Fig. 1). Thus, after this equilibrium is reached (i.e. in the hundreds of a microsecond time range, see Fig. 1), the amount of P+ remaining should be too low to be detectable. Conversely, in the eventual RCs devoid of the tetraheme subunit, P+ is expected to decay via charge recombination, i.e. in the tens of a millisecond time range (23Agalidis I. Sebban P. Biochim. Biophys. Acta. 1995; 1232: 180-186Crossref PubMed Scopus (4) Google Scholar). Thus, the amount of P+ still detectable at 200 μs after the actinic flash can be taken as an indication of the amount of RCs with no tetraheme subunit. Moreover, the absorption changes measured 200 μs after the flash were indicative of the flash-induced oxidation of a low potential heme (data not shown). We take these results as evidence that most of the RCs (97%) have a bound and functional cytochrome subunit. Another possible explanation of the different titration curves in the VC-F and WT RCs is a structural modification of the protein matrix around the bacteriochlorophyll dimer resulting from the chimeric association with the B. viridis cytochrome subunit. We consider this hypothesis as unlikely as well, because the absorption changes associated with the formation of the P+ state in the VC-F mutant were similar to those observed with the WT R. gelatinosus RC (Fig. 4A). Further, EPR measurements performed on VC-F-oriented membrane multilayer dried on Mylar sheets showed an angle dependence of the gz signal of the hemes identical to that obtained for B. viridis. Nitschke and Rutherford (24Nitschke W. Rutherford A.W. Biochemistry. 1989; 28: 3161-3168Crossref Scopus (62) Google Scholar) assigned the EPR gz signals of each of the four hemes in the B. viridis cytochrome on the basis of their respective Em. They ascribed the g-value of 3.09 to the highest potential c559 heme and g-values comprised between 3.29 and 3.32 to the other hemes. The global binding of the B. viridis cytochrome onto the RC of R. gelatinosus was investigated in the same way. Oriented multilayers of VC-F membrane fragments exhibited peaks at g-values of 3.1 and 3.3 with orientation-dependent intensities (Fig. 4B). Polar plots of EPR signal amplitudes allowed one to orient the gz of 3.1 along the 0° axis, whereas the maximum of the gz = 3.3 line was found at 45° (Fig. 4C). Since the gz vector of a heme is perpendicular to the porphyrin ring, we concluded that the highest potential heme c559 was almost perpendicular (90°) to the membrane plane, similar to B. viridis (compare Fig. 4C with Fig. 7 in Ref. 24Nitschke W. Rutherford A.W. Biochemistry. 1989; 28: 3161-3168Crossref Scopus (62) Google Scholar). Moreover, the absorption spectra of the four hemes bound to the tetraheme subunit are conserved in the chimeric RC with respect to the WT B. viridis RC (see Fig. 5A for a comparison of absorption changes associated with the oxidation of the c556 and c559 hemes in both strains). Furthermore, the Ems of the three lower potential hemes embedded in the cytochrome subunit are remarkably conserved when compared with those found in B. viridis (14Maki H. Matsuura K. Shimada K. Nagashima K.V.P. J. Biol. Chem. 2003; 278: 3921-3928Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Based on the sensitivity of both the spectroscopic and redox properties of a redox cofactor to any significant alteration of its protein environment, we take these results as a strong indication in favor of a conserved structure.Fig. 5Dark equilibrium redox titration of the high potential hemes of VC-F and B. viridis RC. The absorbance changes were recorded in the α-band of the cytochromes. Panel A, difference absorption spectra of c556 and c559 oxidation with VC-F and B. viridis membrane fragments (symbols and lines, respectively). The c556 and c559 oxidation spectra were obtained by taking the difference between the spectra measured at +320 and +240 mV and measured at +500 and + 400 mV, respectively. Panel B, titration curve of c556 heme at 556–562 nm with VC-F (solid symbols) and B. viridis (open symbols) membrane fragments. Panel C, titration curve of c559 heme at 558–552 nm VC-F (solid symbols) and B. viridis (open symbols) membrane fragments. r.u., relative units.View Large Image Figure ViewerDownload (PPT) As a third hypothesis, which would account for the singular titration curve of the P+/P couple in the VC-F strain, we would now like to consider the existence of a significant electrostatic interaction between the bacteriochlorophyll dimer and its nearest redox-active neighbor, the c559 heme. Such an interaction between two close redox cofactors implies that the Em of either one of the two cofactors depends on the redox state of its neighbor. In the present framework, one should thus consider the following schemes as shown in Equations 1, 2, 3, 4, 5, 2The reduced and oxidized states of the cytochrome are noted as c and c+, respectively, for the sake of clarity. It does not mean that the absolute charge of the reduced cytochrome is null or that it is equal to one for the oxidized state. Yet, the change in charge resulting from the oxidation is equal to one electron.Pc↔EP(c)P+c+e−(Eq. 1) Pc+↔EP(c+)P+c++e−(Eq. 2) Pc+↔EP(c+)P+c++e−(Eq. 3) P+c↔Ec(P+)P+c++e−(Eq. 4) with{EP(c+)=EP(c)+ΔψEc(P+)=Ec(P)+Δψ(Eq. 5) where EP(c+) and EP(c) are the Em values of the P+/P couple in the presence of the oxidized and reduced cytochromes, respectively, Δψ is the electrostatic interaction between both cofactors, and Ec(P+) and Ec(P) are the Em values of the cytochrome in presence of P+ and P, respectively. The equation used to fit the titration curve is then derived from both following points. 1) The total amount of oxidized P+ or of reduced P can be written as shown in Equations 6 and 7.[P+]=[P+c+]+[P+c](Eq. 6) [P]=[Pc+]+[Pc](Eq. 7) 2) Applying the Nernst equation at a given ambient potential (Eh) to the four electron transfer reactions just described yields Equations 8, 9, 10, 11.[P+c][Pc]=10Eh−EP(c)60(Eq. 8) [P+c+][Pc+]=10Eh−EP(c+)60=10Eh−EP(c)−Δψ60(Eq. 9) [Pc+][Pc]=10Eh−Ec(P)60(Eq. 10) [P+c+][P+c]=10Eh−Ec(P+)60=10Eh−Ec(P)−Δψ60(Eq. 11) Incorporating Equations 8, 9, 10, 11 into Equations 6 and 7 at a given Eh, the fraction of P in the oxidized state, irrespective of the redox state of the cytochrome is shown in Equation 12.[P+][P+]+[P]=10Eh−EP(c)60+102Eh−Ec(P)−EP(c)−Δψ601+10Eh−EP(c)60+10Eh−Ec(P)60+102Eh−Ec(P)−EP(c)−Δψ60(Eq. 12) This equation allows one to distinguish several cases, each of the two extreme ones where EP(c) « Ec(P) or Ec(P) « EP(c+) yields a redox titration, which follows a one-electron Nernst curve (with Em = EP(c) and Em = EP(c) +Δψ, respectively). This reflects the fact that the redox changes of each cofactor occur in well separated redox potential ranges; thus, they do not interfere. A third and experimentally more interesting case arises when EP(c) ≈ Ec(P) or Ec(P) ≈ EP(c+), because according to the above equation, the titration curve should be markedly different from a classical Nernst curve. This latter case applies to the VC-F strain. The data presented in Fig. 2 could be satisfyingly fitted to Equation 12 (Fig. 2, solid lines) with Ec(P), EP(c), and Δψ as varying parameters. In the VC-F strain, the best-fit parameters were: Ec(P) = +380 mV; EP(c) = +350 mV; and Δψ = +50 mV. In the R. gelatinosus RC, the fit to Equation 12 yielded Ec(P) =+300 mV, EP(c) =+350 mV, and Δψ=+50 mV. In B. viridis, because the Ems of the two interacting cofactors belong to more distinct redox potential regions, the deviation to a one-electron Nernst curve was, as expected, less pronounced. The data could be satisfyingly fitted either with a one-electron Nernst curve with EP =+500 mV or, according to Equation 12, with Ec(P) =+380 mV, EP(c) =+450 mV, and Δψ=+50 mV. It is noteworthy that the values found for the Em of the P+/P couple as well as for Δψ were similar in the chimeric and "parent" RC from R. gelatinosus. Yet, one could argue that the value found here for the Em of the P+/P couple in R. gelatinosus WT is 50 mV smaller than the previously reported one of 400 mV. This was not unexpected, because in the WT, the Em of the closest heme to P is significantly lower than that of the P+/P couple so that the respective oxidation (or reduction) of the two cofactors occurs in distinct redox potential domains. Consequently, the most significant fraction of P should be oxidized in the presence of the oxidized cytochrome and the titration curve should yield, as a first approximation, a Em of EP(c) + Δψ = 350 + 50 =+400 mV, in good agreement with previous reports (22Dutton P.L. Biochim. Biophys. Acta. 1971; 226: 63-80Crossref PubMed Scopus (347) Google Scholar). To further test this interpretation, we have measured the equilibrium redox titration of the P+/P couple in a site-directed mutant of the VC-F strain in which the Em of the c559 heme is expected to be decreased, thereby increasing the gap between the Ems of P and its closest heme. According to the calculation of Gunner and Honig (7Gunner M.R. Honig B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9151-9155Crossref PubMed Scopus (174) Google Scholar), the presence of a positively charged arginine residue (Arg-264) in the vicinity of the c559 heme of the B. viridis cytochrome subunit contributes to raise the midpoint potential of this redox center. This finding was confirmed by Chen et al. (26Chen I.P. Mathis P. Koepke J. Michel H. Biochemistry. 2000; 39: 3592-3602Crossref PubMed Scopus (38) Google Scholar) who substituted, by site-directed mutagenesis, this residue for a lysine and found a downshifted Em for the c559 by ∼110 mV. We have also targeted this Arg and substituted it for a Leu in the gene coding for the tetraheme subunit in the VC-F strain (17Nagashima K.V.P. Alric J. Matsuura K. Shimada K. Verméglio A. Bruce D. van der Est A. 13th International Congress on Photosynthesis. Allen Press, Montréal2004Google Scholar). Consistent with the previous findings from Chen et al. (26Chen I.P. Mathis P. Koepke J. Michel H. Biochemistry. 2000; 39: 3592-3602Crossref PubMed Scopus (38) Google Scholar), the Em of the c559 heme in this mutant (R264L) was decreased to 130 mV (17Nagashima K.V.P. Alric J. Matsuura K. Shimada K. Verméglio A. Bruce D. van der Est A. 13th International Congres
Referência(s)