The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Asp235 in yeast cytochrome c peroxidase forms a hydrogen bond with His175, the proximal histidyl residue, that has been suggested to be important in determining the electronic properties of the heme iron and that may be involved in stabilizing the higher oxidation states of the peroxidase that form transiently during catalysis. The current study employs 1H and 15N-NMR spectroscopy to study the electronic properties of and the effects of pH on the active site of the Asp235Ala variant. This variant exhibits three spectroscopic species between pH 5 and 9: a high-spin species that forms at low pH and two low-spin species that form successively at higher pH. Nevertheless, the activity of the variant exhibits a pH dependence virtually identical to that of the wild-type protein, though the activity of the variant is 3 orders of magnitude lower at all values of pH between pH 5 and 8.5. These findings suggest that the spin state and coordination environment of the heme iron in cytochrome c peroxidase do not dictate the rate of substrate (cytochrome c) oxidation. Binding of cyanide to the variant enzyme results in formation of a single species as detected by NMR spectroscopy. Analysis of high-resolution 1D and 2D 1H-NMR and 15N-NMR spectra of the cyanide adduct has permitted characterization of the properties of this derivative and the strength of the proximal ligand bond to the heme iron. Disruption of the hydrogen bond between the proximal histidine and Asp235 that exists in the wild-type enzyme dramatically reduces the strength of the interaction between the proximal ligand and the iron; this effect combined with concurrent changes in the distal heme-binding pocket accounts for the increase in reduction potential reported for the Fe3+/Fe2+ couple. The catalytic consequences of the structural and electronic properties of the variant elucidated in this study are discussed.

The structural changes in oxidized yeast iso-1-cytochrome c and fully oxidized bovine cytochrome c oxidase that are induced upon complex formation have been analyzed by resonance Raman spectroscopy. The main spectral changes could be ascribed to cytochrome c, which in the case of the wild-type protein are essentially the same as previously observed in the complex of horse heart cytochrome c and bovine cytochrome c oxidase [Hildebrandt et al. (1990) Biochemistry 29, 1661-1668]. These spectral changes are attributed to the formation of the conformational state II (approximately 45%) which exhibits an open heme pocket structure. The structural changes are assumed to be induced by the electrostatic interactions between the negatively charged binding domain on cytochrome c oxidase and the positively charged lysine residues on the front surface of cytochrome c. Substituting one of these lysine residues (i.e., Lys-72) by an alanine significantly lowers the state II content (< 15%), implying that this lysine is essential for controlling the conformational equilibrium of the bound protein. On the other hand, the replacement of lysine-79 by alanine only slightly lowers the state II content (approximately 35%). However, the analysis of the spectra suggests that lysine-79 may be involved in controlling conformational details within the heme pocket of the bound cytochrome c. Due to the underlying structural changes and the lowered redox potential, formation of state II may be of functional importance for the physiological electron-transfer process by lowering the reorganization energy and increasing the driving force. The spectral changes caused by complex formation that are attributable to cytochrome c oxidase indicate structural changes of the vinyl and formyl substituents while the ground-state conformations of the porphyrin macrocycles are preserved. This finding implies that the conformational changes in the heme pockets of cytochrome c oxidase are much smaller than those in cytochrome c. These changes refer not only to heme a but also to heme a3, located remote from the cytochrome c binding site, pointing to a long-range structural communication between the binding domain and the oxygen reduction site. The possible functional implications of these structural changes are discussed.