Models for the complexes formed between cytochrome b5 and the subunits of methemoglobin

Computer graphics-generated models for the electron transfer complexes formed between cytochrome b5 and the subunits of methemoglobin are proposed. For both complexes, the orientation allowing optimal hydrogen bonding involves interaction between negatively charged residues on cytochrome b5 and positively charged residues on methemoglobin. In each complex, the heme groups of the interacting species are coplanar with the edges of the heme groups separated by 7-8 A and with the iron atoms 16 A apart. For the alpha-chain X cytochrome b5 complex, alpha-chain residues 56 (Lys), 60 (Lys), and 90 (Lys) interact with cytochrome b5 residues 44 (Glu), 43 (Glu), and 60 (Asp) respectively. A fourth hydrogen bond involves alpha-61 (Lys) bridging between a heme propionate from cytochrome b5 and a heme propionate from the alpha-chain. The contacts present in the beta-chain X cytochrome b5 complex involve hydrogen-bonding between beta-chain lysyl residues 59, 61, 65, and 95, and cytochrome b5 residues 48 (Glu), 44 (Glu), 43 (Glu), and 60 (Asp) respectively. An additional hydrogen bond can be formed by bridging of the epsilon-amino group of beta-66 (Lys) between a heme propionate from cytochrome b5 and a beta-chain heme propionate. In each complex, two nonionic interactions, one on each side of the heme groups, are also suggested. These interactions appear to effectively exclude external water molecules from the center of the protein-protein interaction domain. Comparison of the proposed binding loci for cytochrome b5 on the methemoglobin subunits with those proposed on cytochrome c reveals considerable structural homology between the cytochrome b5 binding sites.     

The Regulation of Surface Charged Residues on the Properties of Cytochrome b5

To understand the roles of negatively surface charged residues, the cytochrome b₅ (Cyt b₅) E48A/D60A mutant was constructed. UV-visible and CD spectra confirmed that the mutation did not cause overall structural changes of the protein. The mutant presents an unexpected high stability toward the thermal and denaturant compared with the wild type Cyt b₅, which shows that these surface charged residues can influence the interactions between the heme b group and the polypeptide chain. Functional properties were clarified through the electron transfer reactions between Cyt b₅ and Cyt c. The driving force of the electron transfer reactions is conservative. Although the association constant of Cyt b₅ E48A/D60A with Cyt c is much lower than that of the wild type Cyt b₅, their electron transfer rate constants do not differ significantly. The results show that these surface charged residues play important roles in regulating both the stability and functional properties of Cyt b₅.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

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.     

Spectroelectrochemistry of cytochrome b559 in the D1-D2-Cyt b559 complex from spinach

The redox potential of cytochrome b559 (Cyt b559) in the D1-D2-Cyt b559 complex from spinach has been determined to be +90+/-2mV vs. SHE at pH 6.0, by thin-layer cell spectroelectrochemistry for the first time. The redox potential, corresponding uniquely to the so-called "low-potential form", exhibited a sigmoidal pH-dependence from pH 4.0 to 9.0, ranging from +115 to +50mV. An analysis of the pH-dependence based on model equations suggests that two histidine residues coordinating to the heme iron in the protein subunits may exert electrostatic influence on the redox potential of Cyt b559.     

Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.     

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.     

Kinetic studies on the oxidation of cytochrome b 5 Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.     

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236?±?3?mV in the absence of PEG, which was negatively shifted to +200?±?4?mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0?Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.     

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.     

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. III. Prediction of the docked complex by a complete, systematic search

The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochrome c heme edge is within 4 A of the indole ring of subunit II residue Trp(104), indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys(13) with Asp(119) and Lys(72) with Gln(103) and Asp(158) are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp(104) in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover.     

Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase

Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

The specificity in the interaction between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 is mainly determined by the copper protein

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.     

X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35-->Tyr mutant.

Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site-directed mutagenesis we have produced three mutants of cyt b5: Phe35-->Tyr, Phe35-->Leu, and Phe35-->His. We found that of these three mutants, the Phe35-->Tyr mutant displays abnormal properties. The redox potential of the Phe35-->Tyr mutant is 66 mV more negative than that of the wild-type cyt b5 and the oxidized Phe35-->Tyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. In this study we studied the most interesting mutant, Phe35-->Tyr, by X-ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35-->Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35-->Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.     

A polypeptide chain-refolding event occurs in the Gly82 variant of yeast iso-1-cytochrome c

The replacement of Phe82 in yeast iso-1-cytochrome c by a glycine residue substantially alters both the tertiary structure and electron transfer properties of this protein. The largest structural change involves a polypeptide chain refolding of residues 79 through 85. Refolding places glycines 82, 83 and 84 immediately adjacent to the plane of the heme group in a spatial positioning comparable to that of the phenyl ring of Phe82 in the wild-type protein. Despite this perturbation in structure, solvent accessibility computations show that heme solvent exposure has not increased in the Gly82 variant protein. However, refolding does result in the introduction of a number of polar groups into the hydrophobic heme pocket. This appears to be responsible for the decreased reduction potential of the heme in this protein. The present study, along with that of the Ser82 variant protein (Louie et al., 1988b), clearly establishes the link between dielectric constant within the heme crevice and reduction potential. The further anomalously low electron transfer activity of the Gly82 variant protein would appear to arise from two factors. First, the polypeptide chain medium now adjacent to the heme is unable to facilitate electron transfer in a manner similar to that of the aromatic side-chain of Phe82. Second, polypeptide chain refolding significantly alters the surface contour of the Gly82 protein rendering it less suitable to interact with the corresponding complementary surfaces of redox partners. Our data support the conclusion that Phe82 plays a number of roles in the electron transfer process mediated by yeast iso-1-cytochrome c. These include the maintenance of the heme environment, provision of an optimal medium along the path of electron transfer and formation of interactions at the contact interface in complexes with redox partners.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

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.     

Redox protein electron-transfer mechanisms: electrostatic interactions as a determinant of reaction site in c-type cytochromes

The effect of ionic strength on the rate constant for electron transfer has been used to determine the magnitude and charge sign of the net electrostatic potential which exists in close proximity to the sites of electron transfer on various c-type cytochromes. The negatively charged ferricyanide ion preferentially reacts at the positively charged exposed heme edge region on the front side of horse cytochrome c and Paracoccus cytochrome c2. In contrast, at low ionic strength, the positively charged cobalt phenanthroline ion interacts with the negatively charged back side of cytochrome c2, and at high ionic strength at a positively charged site on the front side of the cytochrome. With horse cytochrome c, over the ionic strength range studied, cobalt phenanthroline reacts only at a positively charged site which is probably not at the heme edge. These inorganic oxidants do not react at the relatively uncharged exposed heme edge sites on Azotobacter cytochrome c5 and Pseudomonas cytochrome c-551, but rather at a negatively charged site which is away from the heme edge. The results demonstrate that at least two electron-transferring sites on a single cytochrome can be functional, depending on the redox reactant used and the ionic strength. Electrostatic interactions between charge distributions on the cytochrome surface and the other reactant, or interactions involving uncharged regions on the protein(s), are critical in determining the preferred sites of electron transfer and reaction rate constants. When unfavorable electrostatic effects occur at a site near the redox center, less optimal sites at a greater distance can become kinetically important.