Reversible carbon monoxide binding has been used to examine the structural and functional properties of reduced Rhodospirillum molischianum cytochrome c'. The symmetrical dimer is found to bind CO in a noncooperative manner, indicating that the heme sites function independently and with identical carbon monoxide affinity. The enthalpy change of binding CO (aqueous) to R. molischianum ferrocytochrome c' is determined to be -11 kcal/mol of CO, which is comparable to the heat of CO binding to other heme proteins. A Bohr effect is observed (0.31 +/- 0.04 proton released per mole of CO bound at pH 8), and a basic group is involved which changes its pK from 8.3 to 7.8 upon ligation. The histidine axial ligand to the heme iron is suggested to be the source of the Bohr effect. Increased CO affinities were observed at high pH or at neutral pH in the presence of phosphate. These solvent-induced changes in CO affinity do not appear to be caused by changes in quaternary structure but rather are more likely brought about by localized changes in the vicinity of the solvent-exposed heme face.