Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

The interaction of borate ions with cytochrome c surface sites: a molecular dynamics study.

Ionic interactions of cytochrome c play an important role in the electron transfer process. Molecular dynamics simulations of the binding of borate ion, which serves as a model ion, at three different cytochrome c surface sites are performed. This work is motivated by previous NMR studies of cytochrome c in borate solution, which indicate the existence of two types of binding sites, a slow exchange site and a fast exchange site. These two types of binding behavior were observed in the dynamic simulations, offering a molecular interpretation of "loose" and "tight" binding. At the "loose" binding sites (near Lys25/Lys27 and Lys55/Lys73) the ion forms two to three hydrogen bonds to the nearest lysine residue. This binding is transient on the time scale of the simulation, demonstrating the feasibility of fast exchange. At the "tight" binding site (near Lys13/Lys86), on the other hand, the ion becomes integrated into the protein hydrogen bond network and remains there for the duration of the simulation (exemplifying slow exchange). Binding simulations of the ion at the "tight" site of H26Q mutant cytochrome c also showed integration of the ion into the protein's hydrogen bond network. However, this integration differs in details from the binding of the ion to the native protein, in agreement with previous NMR observations.     

Location of a cytochrome c binding site on the surface of flavocytochrome b 2

Saccharomyces cerevisiae flavocytochrome b ₂ couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate constant for cytochrome c reduction by flavocytochrome b ₂ depends on the rate of complex formation and is sensitive to ionic strength. Mutations in the heme domain of flavocytochrome b ₂ (Glu63→Lys, Asp72→Lys and the double mutation Glu63→Lys:Asp72→Lys) have significant effects on the reaction with cytochrome c, implicating these residues in complex formation. This kinetic information has been used to guide molecular modelling studies, which are consistent with there being no one single best-configuration. Rather, there is a set of possible complexes in which the docking-face of cytochrome c can approach flavocytochrome b ₂ in a variety of orientations. Four cytochromes c can be accommodated on the flavocytochrome b ₂ tetramer, with each cytochrome c forming interactions with only one flavocytochrome b ₂ subunit. All the models involve residues 72 and 63 on flavocytochrome b ₂ but in addition predict that Glu237 may also be important for complex formation. These acidic residues interact with the basic residues 13, 27 and 79 on cytochrome c. Through this triangle of interactions runs a possible σ-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavocytochrome b ₂) and ends with the ring of Pro68, which is in van der Waals contact with the cytochrome c heme. In total, the edge-to-edge "through space" distance from the imidazole ring of His66 to the C3C pyrrole ring of cytochrome c is 13.1?Å.     

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

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.     

Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene

Deletion of the cytochrome c2 gene in the purple bacterium Rhodobacter sphaeroides renders it incapable of phototrophic growth (strain cycA65). However, suppressor mutants which restore the ability to grow phototrophically are obtained at relatively high frequency (1-10 in 10(7)). We examined two such suppressors (strains cycA65R5 and cycA65R7) and found the expected complement of electron transfer proteins minus cytochrome c2: SHP, c', c551.5, and c554. Instead of cytochrome c2 which elutes from DEAE-cellulose between SHP and cytochrome c', at about 50 mM ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c551.5 at about 150 mM ionic strength. The new cytochrome is more acidic than cytochrome c2, but is about the same size or slightly smaller (13,500 Da). The redox potential of the new cytochrome from strain cycA65R7 (294 mV) is about 70 mV lower than that of cytochrome c2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c2, which suggests that there is less tryptophan (the latter has two residues). In vitro kinetics of reduction by lumiflavin and FMN semiquinones show that the reactivity of the new cytochrome is similar to that of cytochrome c2, and that there is a relatively large positive charge (+2.6) at the site of reduction, despite the overall negative charge of the protein. This behavior is characteristic of cytochromes c2 and unlike the majority of bacterial cytochromes examined. Fourteen out of twenty-four of the N-terminal amino acids of the new cytochrome are identical to the sequence of cytochrome c2. The N-termini of the cycA65R5 and cycA65R7 cytochromes were the same. The kinetics and sequence data indicate that the new protein may be a cytochrome c2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c2 for the new cytochrome produced in the suppressor strains.     

Cocrystals of yeast cytochrome c peroxidase and horse heart cytochrome c

Yeast cytochrome c peroxidase and horse heart cytochrome c have been cocrystallized in a form suitable for x-ray diffraction studies and the structure determined at 3.3 A. The asymmetric unit contains a dimer of the peroxidase which was oriented and positioned in the unit cell using molecular replacement techniques. Similar attempts to locate the cytochrome c molecules were unsuccessful. The peroxidase dimer model was subjected to eight rounds of restrained parameters least squares refinement after which the crystallographic R factor was 0.27 at 3.3 A. Examination of a 2Fo-Fc electron density map showed large "empty" regions between peroxidase dimers with no indication of cytochrome c molecules. Electrophoretic analysis of the crystals demonstrated the presence of the peroxidase and cytochrome c in an approximate equal molar ratio. Therefore, while cytochrome c molecules are present in the unit cell they are orientationally disordered and occupy the space between peroxidase dimers.     

Recombinant cytochrome c peroxidase folds properly without conformational "annealing".

Recombinant cytochrome c peroxidase isolated from Escherichia coli has recently been reported to exhibit an abnormal electronic absorption spectrum that is converted to the normal spectrum after conformational "annealing" of the recombinant enzyme by passage over a cytochrome c affinity column. The current report provides evidence that the abnormal spectrum observed in some preparations of recombinant cytochrome c peroxidase arises from the presence of contaminant, damaged forms cytochrome c peroxidase with altered spectra. Removal of these contaminant forms produces a major cytochrome c peroxidase fraction with a normal spectrum. We conclude that elution of recombinant cytochrome c peroxidase over a cytochrome c affinity column does not produce normal enzyme through conformational "annealing" but that it produces purified enzyme through removal of contaminants.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 greater than Lys 25 congruent to Lys 79 approximately equal to Lys 87 greater than Lys 8 approximately equal to Lys 27 approximately equal to Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

Electron transfer between cytochrome c2 and the tetraheme cytochrome c in Rhodopseudomonas viridis

Kinetics of electron transfer from soluble cytochrome c₂ to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome -bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c₂ present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c₂, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c₂ and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c₂ is 60 ± 20 s. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c₂ to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 10⁷ M^-1 s^-1 to 1.3 × 10⁶ M^-1 s^-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 10⁷ M^-1 s^-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c₂ with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c₂ docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.     

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.     

Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR

Hydrogen exchange and two-dimensional nuclear magnetic resonance (2D NMR) techniques were used to characterize the structure of oxidized horse cytochrome c at acid pH and high ionic strength. Under these conditions, cytochrome c is known to assume a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. In order to measure the rate of hydrogen-deuterium exchange for individual backbone amide protons in the A state, samples of oxidized cytochrome c were incubated at 20 degrees C in D2O buffer (pD 2.2, 1.5 M NaCl) for time periods ranging from 2 min to 500 h. The exchange reaction was then quenched by transferring the protein to native conditions (pD 5.3). The extent of exchange for 44 amide protons trapped in the refolded protein was measured by 2D NMR spectroscopy. The results show that this approach can provide detailed information on H-bonded secondary and tertiary structure in partially folded equilibrium forms of a protein. All of the slowly exchanging amide protons in the three major helices of native cytochrome c are strongly protected from exchange at acid pH, indicating that the A state contains native-like elements of helical secondary structure. By contrast, a number of amide protons involved in irregular tertiary H-bonds of the native structure (Gly37, Arg38, Gln42, Ile57, Lys79, and Met80) are only marginally protected in the A state, indicating that these H-bonds are unstable or absent. The H-exchange results suggest that the major helices of cytochrome c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop region of the native structure is flexible and partly disordered.     

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.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)