Methionine Ligand Lability in Bacterial Monoheme Cytochromes c: An Electrochemical Study

Abstract

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 have 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 (Em ~ -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 ± 1 kJ/mol) is similar the energies associated with “foldons” of known protein folding pathways.

Methionine Ligand Lability of Homologous Monoheme Cytochromes c

“…1 Here, we have extended these findings, by both interrogating the nature of the Met-loss state and demonstrating the generality of the conformational change (and the energetics thereof) as monoheme cyts c interact with EPG electrodes.…”

“…1 We concluded that interaction with the EPG surface substantially lowers the free energy for the conformational rearrangement of the Met-donating loop for the Ht cytochrome. 1 Here, we build upon our previous findings of Met-loss behavior by extending our characterization of bacterial monoheme cytochromes c, and we demonstrate that the phenomenon is general to Met-/Hisligated cytochromes c. We show that the relative propensity for loss of the Met-ligand can be directly correlated to the global thermal stability of the cyt c of interest, though not the apparent stability of the Fe−Met bond alone. Finally, we find that the pK a values for the Fe (II/III) redox couple for the various Met-loss states are all largely similar, showing a distinct pattern of H + -coupling that evokes other precedents of bis-His-ligated cyts c, yet may not unambiguously implicate a specific misligated state.…”

“…Finally, we are able to compare the relative populations of the normal and Met-loss states on the electrode to elucidate the Gibbs free energy for the conformational change. 1 Bolstering these data with the observation of the global unfolding temperatures, and relative stabilities of the Fe-Met bond (inferred via the temperature stability of the Met to Fe chargetransfer band at 695 nm) for each cyt c paint a picture where Met-ligand lability occurs readily among monoheme cyts c when bound at EPG electrodes. Taken together, we demonstrate an overall correlation between the spontaneity of the conformational transformation and global folding stability.…”

Kinetics of Oxygen Reduction by a Beta Barrel Heme Protein on Hyrid Bioelectrodes

“…Instead, the unfolded state of these proteins is characterized by only a low potential signal, which, in some proteins was similar to the folded Met‐loss form while differing in others depending on the protein source. The Met61Ala mutant in Hydrogenobacter thermophilus (Ht) cyt c , expected to produce a His/aqua coordination environment without the axial Met ligand, showed a single low potential peak at −163 mV, which increased to −69 mV in the imidazole‐bound form, where the external imidazole was expected to occupy the 6th coordination position of the heme …”

Investigation of the Key Heme in Cytchrome <i>c</i>₃ to the Electron Pool Effect by Highly Sensitive EQCM Technique

“…The four hemes have two histidine as an axial ligand, so that redox potential of the hemes is low compared with Met-Fe-His heme, such as cytochrome c. 15 Thus, cytochrome c 3 has multiple redox states caused by four hemes redox, and the electron pool effect works during the four hemes redox.…”