Effects of pH on protein association: Modification of the proton-linkage model and experimental verification of the modified model in the case of cytochrome c and plastocyanin

Effects of pH on protein association are not well understood. To understand them better, we combine kinetic experiments, calculations of electrostatic properties, and a new theoretical treatment of pH effects. The familiar pr oton-linkage model, when used to analyze the dependence of the association constant K on pH, reveals little about the individual proteins. We modified this model to allow determination not only of the numbers of the H+ ions i nvolved in the association but also of the pK(a) values, in both the separa te and the associated proteins, of the side chains that are responsible for the dependence of K on pH. Some of these side chains have very similar pK( a) values, and we treat them as a group having a composite pK(a) value. Use of these composite pK(a) values greatly reduces the number of parameters a nd allows meaningful interpretation of the experimental results. We experim entally determined. the variation of K in the interval 5.4 less than or equ al to pH less than or equal to 9.0 for four diprotein complexes, those that the wild-type cytochrome c forms with the wild-type plastocyanin and its m utants Asp42Asn, Glu59Gln, and Glu60Gln. The excellent fittings of the expe rimental results to the modified model verified this model and revealed som e unexpected and important properties of these prototypical redox metallopr oteins. Protein association causes a decrease in the pKa values of the acid ic side chains and an increase in the pK(a) values of the basic side chains . Upon association, three carboxylic side chains in wild-type plastocyanin each release a H+ ion. These side chains in free plastocyanin have an anoma lously high composite pK(a) value, similar to6.3. Upon association, five or six side chains in cytochrome c, likely those of lysine, each take up a H ion. Some of these side chains have anomalously low pK(a) values, less tha n 7.0. The unusual pK(a) values of the residues in the recognition patches of plastocyanin and cytochrome c may be significant for the biological, fun ctions of these proteins. Although each mutation in plastocyanin markedly, and differently, changed the dependence of K on pH, the model consistently gave excellent fittings. They showed decreased numbers of H+ ions released or taken up upon protein association and altered composite pK(a) values of the relevant side chains. Comparisons of the fitted composite pK(a) values with the theoretically calculated pK(a) values for plastocyanin indicated t hat Glu59 and Asp61 in the wild-type plastocyanin each release a H+ ion upo n association with cytochrome c. Information of this kind cannot readily be obtained by spectroscopic methods. Our modification of the proton-linkage model is a general one, applicable also to ligands. other than H+ ion and t o processes other than association.