Methionine Ligand Lability of Homologous Monoheme Cytochromes c

Abstract

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

Semisynthetic and Biomolecular Hydrogen Evolution Catalysts

“…Mercury is not generally a biocompatible electrode because of its hydrophobicity, although it was utilized here for its large cathodic window and to facilitate comparison to published work . Future studies will make use of more biocompatible electrodes such as pyrolytic graphic edge electrodes. − However, previous work has shown that, even on pyrolytic graphite, cytochrome c can occupy more than one conformation, and thus perturbation of the protein structure by surface binding remains a concern. , Another approach underway is the activation of Ht -CoM61A by pairing it with photosensitizers for photoinduced electron transfer. In addition to preserving the structure and fold of the protein and its variants, an advantage of this approach is that it accomplishes the storage of light energy in the form of H 2 .…”

“…100−102 However, previous work has shown that, even on pyrolytic graphite, cytochrome c can occupy more than one conformation, and thus perturbation of the protein structure by surface binding remains a concern. 103,104 Another approach underway is the activation of Ht-CoM61A by pairing it with photosensitizers for photoinduced electron transfer. In addition to preserving the structure and fold of the protein and its variants, an advantage of this approach is that it accomplishes the storage of light energy in the form of H 2 .…”

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

“…Films generated using oxidized (“closed”) enzyme could not be converted to the open state by poising at highly oxidizing potentials (>700 mV vs SHE). Previous observations have suggested that the highly polar graphite surface can select for certain conformations of enzymes or promote unfolding of His/Met cytochromes. − In the case of So CcP, the “closed” to “open” conformational shift presumably results in the motion of surface loops rich in both positively and negatively charged amino acid side chains. It is likely that these differing charge distributions account for different affinities to the graphite surface.…”

Maximizing (Electro)catalytic CO2 Reduction with a Ferredoxin-Based Reduction Potential Gradient

“…oneidensis (SoC5) that has a defined absorption difference between its oxidized and reduced state . SoC5 has an E m of ∼310 mV under the assay conditions (pH 7.0) and serves as the electron acceptor for Fds. As the native electron acceptor for DaPFOR, the apparent K M determined for DaFdI (86 nM) is much lower than that determined for HtFd1 (5.1 μM) (Figure S7).…”

“…In the biochemical assay, the reduction of Fd was coupled to a downstream reaction that contains a better chromophore (Figure S1A), in this case, the cytochrome c 553 from S. oneidensis (SoC5) that has a defined absorption difference between its oxidized and reduced state. 43 SoC5 has an E m of ∼310 mV under the assay conditions (pH 7.0) 76 and serves as the electron acceptor for Fds. As the native electron acceptor for DaPFOR, the apparent K M determined for DaFdI (86 nM) is much lower than that determined for HtFd1 (5.1 μM) (Figure S7).…”