title: Mutagenesis of the conserved lysine 14 of cytochrome c-550 from Thiobacillus versutus affects the protein structure and the electron self-exchange rate.
abstract: The lysine residue K14 of cytochrome c-550 of Thiobacillus versutus has been mutated to a glutamine (Q) and a glutamate (E) residue. These mutations have a minimal effect on the pKa for replacement of the methionine ligand (the "alkaline transition"), indicating that a presumptive salt bridge between K14 and E11 does not help stabilize the native form. This is in contrast with mitochondrial cytochrome c, where the homologous K13 forms a structurally important salt bridge with glutamate 90. The NMR signals of protons close to the heme iron in wild-type and mutant ferricytochrome c-550 shift considerably with increasing ionic strength. These effects resemble those seen in mitochondrial cytochrome c upon addition of salt and upon complex formation with redox partners. It is likely that electrostatic screening of positive charges near the heme crevice leads to a slight redistribution of the electron density in the heme. At low ionic strength the NMR spectrum of wild-type cytochrome c-550 shows broad peaks. Line widths decrease upon addition of salt up to 200 mM. In K14Q and K14E cytochrome c-550 the line widths are much smaller at low ionic strength. Wild-type cytochrome c-550 may exist in two exchanging conformations, one of which may represent a more open (non-native) form, in analogy with cytochrome c. However, in the case of cytochrome c-550 this non-native form does not show ligand replacement. The electron self-exchange rates of wild type and mutants have been determined as a function of the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Gorlaeus Laboratories, Department of Chemistry, Leiden University, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Ubbink
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
mesh_terms: D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004583:Electrons; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D005973:Glutamine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011487:Protein Conformation; D013855:Thiobacillus / Q000201:enzymology*
chemical_list: D003574:Cytochrome c Group; D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; C031732:cytochrome C-550; D008239:Lysine