Properties of Modified Cytochromes

SUMMARY Reduced (Fe"+) carboxymethylated cytochrome c (Cmcytochrome c) reversibly binds ligands of ferrous iron, e.g. CO, cyanide, ethyl isocyanide, and oxygen, at neutral PH. Titrations of the reduced protein with CO show that over the pH range 6 to 9.5 the stoichiometry of binding is one, and that the spectrum of the Cm-cytochrome c complex with CO is practically pH-independent. The kinetics of CO binding of Cm-cytochrome c have been studied as a function of pH. At low pH values the binding process as observed by stopped flow or photolysis techniques conforms to a simple bimolecular process (2' = 1.6 X 106M-r s-l), whereas at high pH the process, although remaining simple, it is NlOO-fold slower. At intermediate pH values, the binding of CO occurs in a biphasic reaction, the proportions of the fast and slow phases depending on the pH, on the monitoring wave length, and, in the case of photolytic experiments, on the CO concentration. The dissociation rate constants of CO from Cm-cytochrome c were measured by replacement with oxygen or ethyl isocyanide. A model is proposed in which the acid and alkaline conformers of the ligand-free protein have different ligandbinding properties. It is suggested that there is a pH-dependent transition in the CO adduct of Cm-cytochrome c and the pK of this transition is reported. The model is consistent with simple thermodynamic considerations. Binding between ferrous Cm-cytochrome c and 02 has been examined. At pH 6, a stable O2 complex, similar to myoglobin, is formed, whereas at pH 9 0% rapidly oxidized the heme iron

Oxidation-reduction potentials of D-amino acid oxidase.

Abstract This paper reports a study of the oxidation-reduction equilibrium of d-amino acid oxidase, a flavoprotein containing FAD. The oxidation-reduction potential at 50% oxidation (E½) is -0.004 volt at pH 7.0 and 20°, and therefore about 180 mv higher than that of the free coenzyme (FAD). This difference in oxidation-reduction potential may be described in terms of relative affinity of the apoenzyme for the reduced and oxidized forms of the coenzyme. On this basis the affinity constant for the binding of reduced FAD to the apoenzyme is about 106 higher than that of oxidized FAD. The curve relating E½ to pH is in the alkaline range consistent with a slope of about -0.058 volt per pH unit which corresponds to the difference of 1 proton between the oxidized and reduced forms of the enzyme. The apparent pK of the oxidation-linke group, which belongs to the oxidized form, is ∼7.1. The shape of the oxidation-reduction equilibrium curve of d-amino acid oxidase is pH dependent, the value of n increasing from about 1 at pH 8.6 to about 3, or more, at pH 6.6. Under these conditions, therefore, one must consider the existence of functional homotropic interactions between at least 2 FAD molecules. The pH dependence of the cooperative oxidation-reduction equilibrium is discussed in the framework of the theory of linked functions