Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA.
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.
Hederstedt
Lars
L
Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden. Lars.Hederstedt@cob.lu.se
Lewin
Anna
A
Throne-Holst
Mimmi
M
D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015870:Gene Expression; D023061:Gene Order; D005798:Genes, Bacterial; D006418:Heme / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D017384:Sequence Deletion; D013057:Spectrum Analysis
D001426:Bacterial Proteins; C090098:CtaA protein, bacteria; D003573:Cytochrome b Group; D008565:Membrane Proteins; C069808:heme O; C027728:heme a; D006418:Heme; D006639:Histidine