Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His(6)-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K.K., and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287-295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild-type level. On the basis of our findings, we proposed a helical-wheel-projection model of CtaA.