Probing the interactions between component B and the hydroxylase of methane monooxygenase

B. J. Wallar, John D Lipscomb

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The sohJble methane nl,Jnooxygenase (MMO) enzyme syslem ccmsists o['three prol(qa components: a hydroxylase (MMOH)lhat conlains a bis-/x hydroxo bridged binuclear iron cluster, a reductase (MMOt/) which serves a() facilitate transfer of electrons to the diiron site, and a B component proaoin (MMOB) containing no known co-fac(ors. The binding of conq)onent tl to MM()]I has been shown to change lhe conformation of the diiron sile, which is maiai['('sled by greatly enhancing the initial velocity, a shift of the redox potential of MMOII. and a dramatic increase in the rat(' of 02 binding and activation by MMOH. The binding interaction between MMOH and MMOB has been examined more closely. The component B gene has been snbch)ned and owarexpressed into E. coli and recombinant protein can easily be puritied. As MMOB contains no cysteine residues, site-directed mulagenesis has been perfl)rmed to generate an accessible cysteine, which was subsequently labeled with sulfhydryl-specific fluorescent labels. These labels hawa proven useful in pro riding a specific spectroscopic handle for the MMOII-MMOB interaction whih, testing pt{, salt. detergent, and component effects. The results conlirm lhal MMOB forms a tight complex with MMOH at physiological conditions, l)y namic and thermodynamic characterization of the complex using the labeled MMOB provides insight into the mechanism by which MMOB exerts its varied (affa,cts. This work was supported by NIl{ grants (;M-l()16fi and GM-07323.

Original languageEnglish (US)
JournalFASEB Journal
Issue number9
StatePublished - Dec 1 1997

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