Methionine ligand lability in bacterial monoheme cytochromes c: an electrochemical study.

The direct electrochemical analysis of adsorbed redox active proteins has proven to be a powerful technique in biophysical chemistry, frequently making use of the electrode material pyrolytic "edge-plane" graphite. However, many heme-bearing proteins such as cytochromes c have been also examined systematically at alkanethiol-modified gold surfaces, and previously we reported the characterization of the redox properties of a series of bacterial cytochromes c in a side-by-side comparison of carbon and gold electrode materials. In our prior findings, we reported an unanticipated, low potential (E(m) ∼ -100 mV vs SHE) redox couple that could be analogously observed when a variety of monoheme cytochromes c are adsorbed onto carbon-based electrodes. Here we demonstrate that our prior phenomological data can be understood quantitatively in the loss of the methionine ligand of the heme iron, using the cytochrome c from Hydrogenbacter thermophilum as a model system. Through the comparison of wild-type protein with M61H and M61A mutants, in direct electrochemical analyses conducted as a function of temperature and exogenous ligand concentration, we are able to show that Met-ligated cytochromes c have a propensity to lose their Met ligand at graphite surfaces, and that energetics of this process (6.3 ± 0.2 kJ/mol) is similar to the energies associated with "foldons" of known protein folding pathways.