Heme A Synthase Enzyme Functions Dissected by Mutagenesis ofBacillus subtilisCtaA

Abstract

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 an affinity 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.

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…The aforementioned heme a synthase (CtaA) is predicted to contain eight TMDs, interestingly, with five conserved histidines that are located on the periplasmic side of the membrane; these, in theory, are for coordinating two hemes that were shown to be present (76). One is a b heme that transfers electrons in the oxidative reaction, and the other is the heme o substrate (32,(149)(150)(151).…”

“…This result is conceptually reminiscent of the heme a synthase encoded by ctaA described above. Recall that CtaA has a heme b which is involved in electron transfer and the heme o substrate for modification to heme a (76). Based on experimentally determined topological models for CcmF (62,129) and new phoA fusion results (125), a unified topological diagram for CcmF is shown in Fig.…”

The Heme a Synthase Cox15 Associates with Cytochrome c Oxidase Assembly Intermediates during Cox1 Maturation

“…Thus, we made use of a previously characterized point mutation in a conserved histidine of the heme a synthase of Bacillus subtilis (CtaA), which displays severely reduced activity (Fig. 3A) (44). Using site-directed mutagenesis, we generated a plasmid-borne mutant, COX15, encoding the protein with a H 368 M exchange.…”

Positively Regulated Glycerol/G3P-Dependent <i>Bacillus subtilis</i> Gene Expression System Based on Anti-Termination

“…The ctaA gene, without its promoter region and engineered to encode CtaA with a C-terminal His 6 -tag, was cloned in pLALA, resulting in pLAL3. As a control plasmid and reference for CtaA protein production levels we used pCTHI10 which contains the ctaA gene with its native promoter region [Hederstedt et al, 2005]. B. subtilis strain LMT20R is deleted for the ctaA gene.…”

“…CtaA was isolated from detergent-solubilized membranes using affinity chromatography and the relative yields of protein were estimated from quantitative Western blots as described in 'Experimental Procedures'. ments [Hederstedt et al, 2005]. The ctaA gene, without its promoter region and engineered to encode CtaA with a C-terminal His 6 -tag, was cloned in pLALA, resulting in pLAL3.…”