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.