title: Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding.
abstract: Cytochrome c(552) from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an alpha-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c(552), which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.
authors.affiliation: Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: E J
authors.initials: EJ
authors.lastname: Tomlinson
authors.forename: S J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003553:Cystine; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D019791:Guanidine; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D003573:Cytochrome b Group; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D006418:Heme; D003553:Cystine; D019791:Guanidine; D000409:Alanine