Chemical modification of the copperA center in cytochrome c oxidase by sodium p-(hydroxymercuri)benzoate

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Abstract
Cytochrome c oxidase contains a Cu ion electron-transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. The reaction of cytochrome oxidase with sodium p-(hydroxymercuri)benzoate (pHMB) was studied and it was found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center that has EPR and near IR absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified Cu center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal ion center in the catalytic cycle of the enzyme. The unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme. Specifically, these observations support the model in which a secondary electron-transfer pathway not involving CuA exists between the cytochrome c and oxygen binding sites which can function at a rate at least 20% of that in the native enzyme.