Characterization of Rhodobacter sphaeroides cytochrome c(2) proteins with altered heme attachment sites.
In c-type cytochromes, heme is attached to the polypeptide via thioether linkages between vinyl groups on the tetrapyrrole ring and cysteine thiols in a CX(2)CH motif. To study the role of the heme-binding site in c-type cytochrome assembly and function, we generated amino acid changes in this region of Rhodobacter sphaeroides cytochrome c(2) ((15)Cys-Gln-Thr-Cys-His(19)). Amino acid substitutions at Cys(15), Cys(18), or His(19) produced mutant proteins that did not support growth via photosynthesis where this electron carrier is required. Many of these changes appeared to slow signal peptide removal, suggesting that heme attachment is coupled to processing of the c-type cytochrome precursor protein. Inserting an alanine between the cysteine ligands (CycA-Ins17A) did not significantly alter the behavior of this protein in vivo and in vitro, suggesting that the existence of 2 residues between cysteine thiols is not essential for heme attachment to a Class I c-type cytochrome like cytochrome c(2).
Ríos-Velázquez
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Department of Bacteriology, University of Wisconsin-Madison, 312 E. B. Fred Hall, 1550 Linden Drive, Madison, Wisconsin 53706, USA.
Cox
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Donohue
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045362:Cytochromes c2; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013053:Spectrophotometry
D003574:Cytochrome c Group; D008024:Ligands; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006418:Heme; D045362:Cytochromes c2; D003545:Cysteine