Transient intermediates of the methane monooxygenase catalytic cycle

S. K. Lee, J. C. Nesheim, J. D. Lipscomb

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Three new intermediates of the catalytic cycle of the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been detected using transient kinetic techniques. MMO consists of hydroxylase (MMOH), reductase, and 'B' (MMOB) components. MMOH contains an oxygen-bridged [Fe(III)·Fe(III)] cluster that catalyzes O2 activation and insertion chemistry. At 4 °C, rapid mixing of O2 with diferrous MMOH ([Fe(II)·Fe(II)]) in the presence of a 2-fold excess of MMOB resulted in loss of the g = 16 EPR signal characteristic of the diferrous cluster at an apparent first order rate of 22 ± 5 s-1 (O2 ≃ 700 μM). Subsequently, an EPR silent, chromophoric (λ(max) = 330 and 430 nm, ε ≃ 7500 M-1 cm-1 at each wavelength) intermediate (compound Q) formed with an average first order rate constant of 1 ± 0.1 s-1 and then decayed at 0.05 ± 0.01 s- 1. Since compound Q formed much more slowly than diferrous MMOH disappeared, at least one other undetected intermediate (compound P) must have formed before compound Q. MMO substrates had little or no effect on the formation rate of compound Q, but they caused the decay rate to increase linearly with the concentration added. The substrates methane, furan, and nitrobenzene caused compound Q decay to occur with second order rate constants of 19,000 M-1 s-1, 9000 M-1 s-1, and 200 M-1 s-1 (± 5%), respectively. When nitrobenzene was used as a substrate, a second chromophoric intermediate (compound T, λ(max) = 325 nm, with a shoulder at 395 nm, ε395 ≃ 6000 M-1 cm-1) formed at the same rate as compound Q decay. Chemical quench studies showed that compound T is an enzyme-product complex that decays with a rate constant of 0.02 ± 0.005 s-1. This rate is approximately the same as k(cat) for nitrobenzene turnover at 4 °C catalyzed by the reconstituted MMO system, suggesting that product release is the rate-limiting step in catalysis. The characteristics of compound Q suggest that it may be the activated form of the enzyme that directly catalyzes substrate oxidation.

Original languageEnglish (US)
Pages (from-to)21569-21577
Number of pages9
JournalJournal of Biological Chemistry
Issue number29
StatePublished - 1993

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