Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.
The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.
Froland
W A
WA
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
Andersson
K K
KK
Lee
S K
SK
Liu
Y
Y
Lipscomb
J D
JD
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006838:Hydrocarbons / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008772:Methylococcaceae / Q000201:enzymology*; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D013379:Substrate Specificity
D006838:Hydrocarbons; D046911:Macromolecular Substances; D010088:Oxidoreductases; D010105:Oxygenases; C021555:methane monooxygenase