title: 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.
authors.affiliation: Department of Chemistry, Boston University , 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Levin
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Walsh
authors.forename: Kristal K
authors.initials: KK
authors.lastname: Sullivan
authors.forename: Kara L
authors.initials: KL
authors.lastname: Bren
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Elliott
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics; D055664:Electrochemical Techniques; D004566:Electrodes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D043370:Nitrosomonas europaea / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D008715:Methionine; D000409:Alanine