Methane Monooxygenase Hydroxylase

Abstract

AbstractSoluble methane monooxygenase (sMMO) catalyzes the conversion of methane and dioxygen to methanol and water. It is a cytoplasmic system of three proteins produced in methanotrophic bacteria grown under conditions of low copper stress. The best characterized sMMO stems fromMethylococcus capsulatus(Bath). Under physiological conditions inM. capsulatus(Bath), three protein components are required for sMMO activity. These included a 251‐kDa hydroxylase (MMOH), which contains the carboxylate‐bridged, dinuclear iron active site; a 16‐kDa regulatory protein known as protein B (MMOB); and a 38.6‐kDa reductase (MMOR) that contains FAD and a Fe2S2center. MMOH has been characterized by several spectroscopic techniques (UV/vis, X‐ray absorption, Mössbauer, EPR, ENDOR, CD, MCD), which established the nature of the dinuclear iron active site.The crystal structure of the MMOH component of sMMO fromM. capsulatus(Bath) has been solved in two crystal forms, one of which was refined to a resolution of 1.7 Å. The enzyme is composed of two copies each of three subunits (α2ß2γ2), and all three subunits are almost completely α‐helical. The active site of each α‐subunit contains one dinuclear iron center, housed in a four‐helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues, as well as by a bridging hydroxide ion, a terminal water molecule, and at 4°C, a bridging acetate ion, which is replaced at −160°C with a bridging water molecule. Comparison of the results of the two crystal forms shows one structural difference, namely, the altered side chain conformation of Leu110 at the active site cavity. It is suggested that this residue serves as one component of a hydrophobic gate controlling the access of substrates to and products from the active site. MMOB and MMOR bind to the α‐ and ß‐subunits of MMOH, respectively. The structure of MMOB has been known from NMR. X‐ray structures of reduced MMOH and of DMSO‐soaked MMOH have been determined. During the turnover, Hperoxoand Q intermediate states could be trapped and structures proposed from density functional theory studies.