Activation and Inhibitor Studies on Methyl-Coenzyme M Reductase and Purification of a New Hydroxylamine Oxidoreductase from Methylomicrobium Album ATCC 33003

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. In order to manage the overproduction of methane in the environment, the exploration of the cellular regulation of MCR activity and catalytic mechanism of MCR is necessary. Two cellular components, A2 and A3a are involved in the activation of MCR in Methanothermobacter thermoautotrophicus. The effect of A2 and A3a on the activation of MCR from Methanothermobacter marburgensis was tested. MCRsilent and A2 were purified to homogeneity. A3a was purified to 70% purity and it might contain an iron-sulfur cluster. With the presence of minimal required components and the electron donor Ti(III) citrate, there was no significant activity detected in MCR from M. marburgensis. Efforts to reconstitute the cluster content of A3a did not result in the higher activity of MCR. It was concluded that the components A2 and A3a are not involved in or are not sufficient for the activation of MCRsilent from M. marburgensis. There are several hypothetical catalytic mechanisms proposed for MCR. The major difference between these mechanisms is the initial interaction between the substrate methyl-coenzyme M and the nickel atom in the MCR active site, being either a metalloorganic nickel-methyl or a nickel-thiolate complex. With EPR and ENDOR spectroscopic techniques, we were able to show the presence of a Ni(III)-CH3 species in MCR after reaction with bromomethane. This is no proof, but the result give plausibility to hypothetical mechanisms that include such an intermediate in the reaction. Additionally, it was shown by both EPR and NMR spectroscopy that incubation of the Ni(III)-CH3 species with the substrate analog coenzyme M resulted in the formation of methyl-coenzyme M, which is the first evidence for reverse methanogenesis as has been proposed to take place in archaea conducting anaerobic methane oxidation. Hydroxylamine Oxidoreductase from the nitrifier Nitrosomonas europaea is a multi-heme containing protein and has been extensively studied. It is known for long that methanotrophs are also able to oxidize ammonia to nitrate. An HAO-like gene was recently found in the genome of the methanotroph, Methylomicrobium album ATCC 33003. A protein that was capable of oxidizing hydroxylamine was purified from this strain and preliminary characterization showed that this enzyme is a heterodimer and does not contain a heme prosthetic group or iron-sulfur cluster. The properties of this new HAO are compared with known HAOs from various microorganisms.