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The electron-transfer rates between gold electrodes and adsorbed cytochromes are compared for native cytochrome c and its mutant (K13A) using two different immobilization strategies. The study links the protein's heme unit to the SAM, thereby short-circuiting the electron tunneling pathway.

Site specific mutation of cytochrome c at residue 30 reduces protein stability while the same done on residue 67 increases stability. A combination of both single mutations produces recovery of the native characteristics of the protein. Urea titration indicate that single mutations increase the exposure of hydrophobic groups to the solvent, the conformational alteration repaired when the two mutations are combined. However, only partial restoration of the general stability of the protein results from double mutations even though the necessary folding pattern is retained.

Examines the enzymatic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase. Preparation and characterization of cysteine mutants of Cc; Purification of 1:1 CcP-Cc covalent complex; Steady-state kinetics of reaction of 1:1 Cc-CcP covalent complex with Cc; Multiple Cc interaction sites in the high-affinity binding domain.

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates. The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome. Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, DeltaS0'Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- --> Met80-Fe2+-His18. For the Phe82His variant mixed redox couple, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'Rxn = -52 J/mol.K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

To probe the details of protein heme interactions, we have developed a Raman difference spectroscopic technique, which allows reliable detection of very small, approximately equal to 0.01 cm-1, frequency differences. When this technique is applied to heme proteins, structural differences in the protein which perturb the porphyrin macrocycle may be examined by obtaining Raman difference data on the porphyrin vibrational modes which are strongly enhanced in the Raman spectrum produced with visible laser excitation. We report here Raman difference spectroscopic data on cytochromes c from 24 species. The differences in the Raman spectrum of the porphyrin between the cytochromes c of any two species are small, confirming that all of the cytochromes we have examined have the same "cytochrome fold". However, many small (0.02-2 cm-1) but systematic differences were detected which indicate structural differences among these proteins. These differences could be classified into three different groups and interpreted in terms of different types of structural variations resulting from specific differences in the amino acid sequences. First, direct interactions between near-heme residues and the porphyrin influence the electron density in the pi orbitals of the porphyrin macrocycle. Second, variation in the residue at position 92, far removed from the heme, affects the frequency of the core-size marker line at 1584 cm-1. Third, the conformation near cysteine 14 affects the shape of the Raman mode which is sensitive to the pyrrole ring substituents (approximately 1313 cm-1). From these data we conclude that there are several ways in which the protein amino acid sequence may regulate the oxidation-reduction potential and several ways in which the sequence can modify the binding site between cytochrome c and its redox partners.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.

For immunochemical and evolutionary reasons we determined the primary structure of cytochrome c from two strains of laboratory mice. Thioacetylthioethane and thioacetylthioglycolic acid were used in addition to conventional reagents for sequence determinations. The sequence was found to be identical with that of the rabbit except for residues 44 and 89 and consistent with the peptide compositional data reported by Hennig (Hennig, B. (1975), Eur. J. Biochem. 55, 167-183). The rat cytochrome c cymotryptic peptides were identical with those of the mouse in amino acid composition and amino-terminal residues. Further, peptide maps of cytochromes c of the guinea pig and two strains of rat indicate that all these animals have the same cytochrome c as the laboratory mouse. It is concluded that rodent cytochromes c are evolutionarily conservative and that there is no evidence for a generation-time effect in cytochrome c evolution.

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.