Loss of either of the two heme-binding cysteines from a class I c-type cytochrome has a surprisingly small effect on physicochemical properties.
Almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a CXXCH motif. The reasons for the covalent attachment are not understood. Reported here is cytoplasmic expression in Escherichia coli of AXXCH and CXXAH variants of cytochrome c(552) from Hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal stability and reduction potential, to the wild type CXXCH protein. In combination with previous work showing that an AXXAH variant of cytochrome c(552) is much less stable than the CXXCH form, it can be concluded that covalent attachment of heme via either of thioether bonds is sufficient to confer considerable stability and that these bonds contribute little to the setting of the reduction potential. The absence of AXXCH or CXXAH heme-binding motifs from bacterial cytochromes c may relate to the coexistence of the assembly pathway with that for formation of disulfide bonds in the bacterial periplasm.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
E J
EJ
Tomlinson
S J
SJ
Ferguson
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001420:Bacteria, Aerobic / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D004926:Escherichia coli; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic
D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; D019791:Guanidine; D003545:Cysteine