Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor

The isocyclic ring of bacteriochlorophyll (BChl) is formed by the conversion of Mg-protoporphyrin monomethyl ester (MPE) to protochlorophyllide (PChlide), Similarities revealed by BLAST searches with the putative anaerobic MPE-cyclase BchE suggested to us that this protein also uses a cobalamin cofactor. We found that vitamin B-12 (B-12)-requiring mutants of the bluE and bluB genes of Rhodobacter capsulatus, grown without B-12, accumulated Mg-porphyrins. Laser desorption/ionization time-of-flight (LDI-TOF) MS and NMR spectroscopy identified them as MPE and its 3-vinyl-8-ethyl (mvMPE) derivative. An in vivo assay was devised for the cyclase converting MPE to PChlide, Cyclase activity in the B-12-dependent mutants required B-12 but not protein synthesis. The following reaction mechanism is proposed for this MPE-cyclase reaction. Adenosylcobalamin forms the adenosyl radical, which leads to withdrawal of a hydrogen atom and formation of the benzylic-type 13(1)-radical of MPE, Withdrawal of an electron gives the 13(1)-cation of MPE, Hydroxyl ion attack on the cation gives 13(1)-hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 13(1)-keto-MPE, its 13(2)-radical, and cyclization to PChlide.