title: Heme A synthase in bacteria depends on one pair of cysteinyls for activity.
abstract: Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O.
authors.affiliation: The Microbiology Group, Department of Biology, Biology Bld. A, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Lewin
authors.affiliation: The Microbiology Group, Department of Biology, Biology Bld. A, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden. Electronic address: Lars.Hederstedt@biol.lu.se.
authors.forename: Lars
authors.initials: L
authors.lastname: Hederstedt
mesh_terms: D041522:Aeropyrum / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C090098:CtaA protein, bacteria; D003573:Cytochrome b Group; D008565:Membrane Proteins; C069808:heme O; C027728:heme a; D006418:Heme; D003545:Cysteine; D010100:Oxygen
keywords: Aeropyrum pernix; Bacillus subtilis; COX15; CtaA; Cytochrome a; Heme biosynthesis; Protein disulfide