No abstract

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the 02-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type 11, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type I1 methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type I1 Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A and 2.7 8, resolution, respectively, both determined at 18 "C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H20), as well as both carboxylate oxygens of Glu a144. This bis-yhydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 "C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the p chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including Da209E, a ligand of one of the irons.

Soluble methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of three components: hydroxylase, reductase, and component B. The active-site diiron cluster of the hydroxylase has been studied with Mossbauer, ENDOR, and EPR spectroscopies. Móssbauer spectra of the oxidized cluster show that the two high-spin irons are antiferromagnetically coupled in accord with our preliminary study (Fox et al. J. Biol. Chem. 1988, 263, 10553-10556). Mossbauer studies also reveal the presence of two cluster conformations at pH 9. The excited-state S = 2 multiple! of the exchange-coupled cluster (Fe3+-Fe3+) gives rise to an integer-spin EPR signal near g = 8; this is the first quantitative study of such a signal from any system. Analysis of the temperature dependence of the g = 8 signal yields J -15 ± 5 cm™1 for the exchange-coupling constant (Htx = /Si-S2). This value is more than 1 order of magnitude smaller than those reported for the oxo-bridged clusters of hemerythrin and Escherichia coli ribonucleotide reductase (Hex = /Si-S2, J = 270 and 220 cm™1, respectively), suggesting that the bridging ligand of the hydroxylase cluster is not an unsubstituted oxygen atom. Móssbauer spectra of the hydroxylase in applied fields of up to 8 T reveal a paramagnetic admixture of a low-lying excited state into the ground singlet. Both the spectral shape and intensity are well represented by assuming that the spin expectation values for the cluster sites increase