A method has been described for measurement of the oxidation-reduction potentials of redox couples in nonaqueous solutions. This method has been applied to a study of the effect of a nonpolar environment on the redox potential of a heme complex. A positive potential difference of some 300 mV is observed for the measured redox potentials by comparison with reported values for the corresponding heme complex in aqueous solution. The proposal is made that the redox potentials of many high-potential cytochromes may be accounted for by a local heme environment of low-dielectric constant, characteristic of nonpolar amino-acid side chains, and that this factor may play a dominant role in the determination of the oxidation-reduction properties of these proteins.The redox potentials of electron transport proteins are essential to their specific functions in biological systems. In hemeproteins, it has generally been accepted that this property is primarily dependent on the particular porphyrin and the protein groups acting as ligands to the heme iron. The imidazole nitrogen of histidine has long been recognized as the fifth ligand to the heme iron in cytochrome c. Recently, x-ray crystallographic analysis (1) of horse-heart ferricytochrome c has established that the sixth ligand is the sulfur of a methionine residue of the protein. This ligand had been suggested earlier by Harbury et al. (2), who demonstrated that complex formation between methionine and a heme peptide from cytochrome c exhibited an absorption spectrum characteristic of the native protein.The redox potential of this complex is (2) some 300 mV lower than that of cytochrome c (3). Likewise, the standard potentials at pH 7.0 of all other heme complexes measured (4) in aqueous solution are much lower than that of cytochrome c. Yet, potentials of still other c-type cytochromes from plants (5), algae (6), and bacteria (7) have been reported that are significantly higher than cytochrome c. Recent studies of these latter cytochromes by high-resolution nuclear magnetic resonance (NMR) spectroscopy suggest that the ligand groups to the heme iron are the same as those in cytochrome c (8). Thus, a fundamental question remains as to the physical basis for the high oxidation-reduction potentials of cytochrome c and other hemeproteins based on a comparison with the potentials of iron-porphyrins in the presence of ligands thought to typify groups found in these proteins. Toward an understanding of this problem, I considered the effect of a nonpolar environment on the redox potential of the heme complex. For this purpose, the redox potential of bis-pyridine mesoheme dimethylester in benzene was determined. The present report describes both a method for measurement of potentials of oxidation-reduction couples A stock solution of ferrimesoheme dimethylester in benzene was prepared at a concentration that gave a change in absorbance greater than 0.3 at the a-absorption maxima after reduction to the bis-pyridine ferromesoheme ester. Stock solutions of 10 mM potassium f...

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The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.

The Soret absorption maxima and extinction coefficients of the CO and NO complexes of horse myoglobin and (NMeIm)protoheme (NMeIm = 1-methylimidazole) have been determined. The partition coefficient N, equal to the ratio P1/2 (CO)/P1/2(NO), has been determined spectrophotometrically for horse myoglobin and (NMeIm)protoheme. P1/2-(NO) values calculated from the partition coefficients are 5.7 x 10(7) mmHg for (NMeIm)protheme and 1.1 x 10(6) mmHg for horse myoglobin. The ratio of P1/2(NO) values for protein and model is 1.9 which is similar to a value of 1.6 reported for the ratio of P1/2(O2) values. These values may be compared to a ratio of 15 for CO binding to protein and model complexes. This different ratio for CO provides further evidence for steric interaction of the bound CO with the protein based on a consideration of the preferred nonlinear geometry of Fe-NO and Fe-O2 and the linear geometry of Fe-CO.

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