Plastocyanin,an electron-transfer protein

Plastocyanin (Pc) is a copper (Cu)-containing blue protein, that functions as a mobile electron carrier between cytochrome (cyt) f and Photosystem 1 (PS1) in oxygenic organisms. The atomic structure is known and can be described as a -barrel with hydrophobic residues in the interior of the protein. To increase the understanding about structure-function relationships, site-directed mutagenesis of Pc has proven to be very useful. Mainly two spectroscopic techniques, optical and EPR spectroscopy, have been used to investigate how the copper-site is affected by different mutations. The redox properties of the mutants have been investigated and factors that affect the reduction potential are discussed. Absorption and EPR spectra and reduction potentials for the surface mutants are similar to those of the corresponding wild-type. However, mutants around the Cu ion affected the mentioned properties. Comparisons are made with other cupredoxins. Five site-directed mutants of spinach Pc, Pc(Leu12His), Pc(Leu15His), Pc(Thr79His), Pc(Lys81His) and Pc(Tyr83His), have been modified by covalent attachment of a photoactive ruthenium (Ru)-complex at the surface-exposed histidine residues. The rates of the internal electron-transfer reactions exhibit an exponential dependence on the metal-to-metal separation with a decay factor of 1.1 A_-1. A reorganization energy for the Cu-to-Ru electron-transfer reaction of 1.2 eV was determined. Interprotein electron-transfer reactions involving genetically modified Pc are described. Ionic-strength and pH dependencies indicated that electrostatic interactions are involved in the complex formation between Pc and PS 1, which was confirmed by mutations in the acidic patches of Pc. A very specific interaction was further verified by replacements of hydrophobic residues. Position 10, 12, 36, 87 and 90 were found to be very important for the formation of an active complex. A comparison between available structures of Pc and cyt c₆, both effective donors to PS 1, is made. The physiological electron donor to Pc, cyt f, is briefly described.     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

Electron transfer between the spinach plastocyanin mutant Leu12His and Photosystem 1

A spinach plastocyanin (Pc) mutant, Pc(Leu12His), has been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the hydrophobic patch in the interaction with Photosystem 1. The mutant has been characterized by optical absorption, EPR spectroscopy and redox titration. The electron transfer to Photosystem 1 was investigated by flash-induced time-resolved absorption measurements at 830 nm. The Pc(Leu12His) mutant showed a major change in the Photosystem 1 kinetics compared to wild-type Pc. In contrast to the biphasic Photosystem 1 reduction observed for the physiological reaction partner, only the slow phase was discerned when Pc(Leu12His) replaced wild-type Pc as the electron donor. The reaction showed a hyperbolic dependence with increasing Pc concentration, saturating at a rate constant value of 2000 s^-1, which is about 10 times slower than the corresponding rate constant for wild-type Pc. Obviously, this position i s critical for a proper reaction. Moreover, the reaction showed a titrating behavior with a pK_a of 6.7. Thus, it appears that both shape and charge of the residue in this position are important. A plausible reaction mechanism for electron transfer between wild-type Pc and Photosystem 1 is discussed. The role of electrostatic interactions may be that of long-range guidance and initial recognition that allow the two proteins to seek a pre-docking configuration(s). Then a short-range rearrangement(s), involving also hydrophobic interactions, forms an optimum docking configuration prior to electron transfer.     

Dynamic interaction of plastocyanin with the cytochrome bf complex

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.     

Functional characterization of the evolutionarily divergent fern plastocyanin.

Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts. The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners. It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns. Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms. The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step. The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation. This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction. Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs. The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.     

Side-chain interactions in the plastocyanin-cytochrome f complex

Cytochrome f and plastocyanin are redox partners in the photosynthetic electron-transfer chain. Electron transfer from cytochrome f to plastocyanin occurs in a specific short-lived complex. To obtain detailed information about the binding interface in this transient complex, the effects of binding on the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measured. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift changes and these protons are located in the hydrophobic patch (including the copper ligands) and the acidic patches of plastocyanin, demonstrating that both areas are part of the interface in the complex. This is consistent with the recently determined structure of the complex [Ubbink, M., Ejdeb?ck, M., Karlsson, B. G., and Bendall, D. S. (1998) Structure 6, 323-335]. The largest chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron transfer occurs via His87 to the copper in plastocyanin and suggest that the hydrophobic patch determines the specificity of the binding. The chemical-shift changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain solvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the metals in both proteins slightly affects the structure of the complex. The chemical-shift mapping is performed on unlabeled proteins, making it an efficient way to analyze effects of mutations on the structure of the complex.     

Ferredoxin-NADP+ reductase uses the same site for the interaction with ferredoxin and flavodoxin

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.     

Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I.

Plastocyanin (Pc) has been modified by site-directed mutagenesis at two separate electron-transfer (ET) sites: Leu-12-Glu at a hydrophobic patch, and Tyr-83-His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu-12-Glu) and increased by 35 mV in Pc(Tyr-83-His). The latter mutant shows a 2-fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu-12-Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.     

Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal complex formation and electron transfer to ferredoxin

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.     

Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c(6) in Synechococcus sp. PCC 7002 in vivo

Cytochrome c(6) donates electrons to photosystem I (PS I) in Synechococcus sp. PCC 7002. In this work, we provide evidence for rapid electron transfer (t(1/2) = 3 micros) from cytochrome c(6) to PS I in this cyanobacterium in vivo, indicating prefixation of the reduced donor protein to the photosystem. We have investigated the cytochrome c(6)-PS I interaction by laser flash-induced spectroscopy of intact and broken cells and by redox titrations of membrane and supernatant fractions. Redox studies revealed the expected membrane-bound cytochrome f, b(6), and b(559) species and two soluble cytochromes with alpha-band absorption peaks of 551 and 553 nm and midpoint potentials of -100 and 370 mV, respectively. The characteristics and the symmetrical alpha-band spectrum of the latter correspond to typical cyanobacterial cytochrome c(6) proteins. Rapid oxidation of cytochrome c(6) by PS I in vivo results in a unique, asymmetric oxidation spectrum, which differs significantly from the spectra obtained for cytochrome c(6) in solution. The basis for the unusual cytochrome c(6) spectrum and possible mechanisms of cytochrome c(6) fixation to PS I are discussed. The occurrence of rapid electron transfer to PS I in cyanobacteria suggests that this mechanism evolved before the endosymbiotic origin of chloroplasts. Its selective advantage may lie in protection against photo-oxidative damage as shown for Chlamydomonas.     

Electrostatic properties of cytochrome f: implications for docking with plastocyanin.

The electrostatic properties of cytochrome f (cyt f), a member of the cytochrome b6f complex and reaction partner with plastocyanin (PC) in photosynthetic electron transport, are qualitatively studied with the goal of determining the mechanism of electron transfer between cyt f and PC. A crystal structure for cyt f was analyzed with the software package GRASP, revealing a large region of positive potential generated by a patch of positively charged residues (including K58, K65, K66, K122, K185, K187, and R209) and reinforced by the iron center of the heme. This positive field attracts the negative charges of the two acidic patches on the mobile electron carrier PC. Three docked complexes are obtained for the two proteins, based on electrostatic or hydrophobic interactions or both and on steric fits by manual docking methods. The first of these three complexes shows strong electrostatic interactions between K187 on cyt f and D44 on PC and between E59 on PC and K58 on cyt f. Two other manually docked complexes are proposed, implicating H87 on PC as the electron-accepting site from the iron center of cyt f through Y1. The second complex maintains the D44/K187 cross-link (but not the E59/K58 link) while increasing hydrophobic interactions between PC and cyt f. Hydrophobic interactions are increased still further in the third complex, whereas the link between K187 on cyt f and D44 on PC is broken. The proposed reaction mechanism, therefore, involves an initial electrostatic docking complex that gives rise to a nonpolar attraction between the regions surrounding H87 on PC and Y1 on cyt f, providing for an electron-transfer active complex.     

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.     

Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119

Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.     

Genetic interactions of the suppressor 2 of zeste region genes

A wide variety of gain of function mutations have been induced in the Posterior Sex Comb (Psc)--Aristapedioid (Arp)--Suppressor 2 of zeste (Su(z)2) region of the second chromosome of Drosophila. This region contains at least three apparently related genes, two of which we have been studying. Psc1 has previously been used to identify Psc as a Pc group gene; however, it is a complex mutation with both gain and loss of function character. We report here that the Pc group character of Psc is not due to a gain of function and presumably reflects the function of the wild-type gene. We also provide evidence for a maternal function for Psc, as well as the neighboring Su(z)2 gene. Su(z)2 does not appear to be a Pc group gene as it does not act in a synergistic fashion with other Pc group genes in promoting posteriorly directed transformations. However, we have found that mutations in Su(z)2 do interact in a variety of interesting ways with mutations in Pc group genes.     

Pseudomonas aeruginosa cytochrome C(551): probing the role of the hydrophobic patch in electron transfer

Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.     

Intramolecular electron-transfer reaction within a diprotein complex of cytochrome c with ferrylmyoglobin modified with diethylenetriaminepentaacetic acid

Horse heart metmyoglobins modified with diethylenetriaminepentaacetic acid, metMb(DTPA)n (n=1, 2, 4, and 5), were characterized by a MALDI-TOF mass spectrometry, amino-acid sequence analysis, and UV-Vis and CD spectroscopies. The DTPA-binding sites on metMb were Lys47, Lys50, Lys87, Lys145, and Lys147 for metMb(DTPA)5, Lys47, Lys87, Lys145, and Lys147 for metMb(DTPA)4, Lys87 and Lys145 for metMb(DTPA)2, and Lys87 for metMbDTPA, respectively. The modified metMb(DTPA)n showed cytochrome c peroxidase-like activity more efficiently than native metMb: metMb(DTPA)5>metMb(DTPA)4>metMb(DTPA)2> metMbDTPA approximately equals native metMb. The first-order rate constants for the reactions of ferrylMb(DTPA)n (n=2, 4, and 5) with reduced cytochrome c [cyt c(II)] were saturated with concentrations of cyt c(II), suggesting that the electron transfer (ET) occurs within a diprotein complex. The intramolecular ET rate constants in the diprotein complex increased with increasing the number of DTPA ions. The reactions of native ferrylMb and ferrylMbDTPA with cyt c(II) obeyed a second-order rate law. A possible ET mechanism is proposed; cyt c(II) binds the DTPA-linked anionic patch around Lys87, Lys145, and Lys147 region of ferrylMb(DTPA)n.     

Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.     

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.     

The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903

Plastocyanin (Pc) and cytochrome c₆ (Cyt) have been purified to homogeneity from the cyanobacterium Pseudanabaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k_obs) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc.     

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies

The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.