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Chemistry of the diiron site of the soluble MMO hydroxylase
MONO- & DINUCLEAR IRON
NO24
CHEMISTRY OF THE DIIRON SITE OF THE SOLUBLE MM0 HYDROXYLASE
P.C. Wilkins,a M. Fontecave,b N. Deighton,c I.D. Podmore,c M.C.R. Symons,c and H. Dalton.a aDepartment of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK bLEDSS, Universiti Joseph Fourier, 38041, Grenoble, France. ‘Department of Chemistry, University of Leicester, Leicester, LEl 7RH, UK Soluble methane monooxygenase, s-MMO, is a three component enzyme system consisting of an hydroxylase, reductase and a small regulatory protein (B). The hydroxylase contains an Fe-O-Fe center which is believed to be the site of enzyme activity. ESR spectroscopy has proved to be a very powerful method in the study of soluble methane monooxygenase from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. There have been reports of from 2 to 7.6 iron atoms per mol of hydroxylase. Titration of apo hydroxylase with metal ions, monitored by ESR, indicates that only two Mn+ are taken up per mol of hydroxylase and any excess metal ion remains free in solution. A mechanism similar to that for cytochrome P-450 has been proposed for the s-MM0 catalysed insertion of oxygen into a C-H bond. Two pathways were suggested - one involving homolytic dioxygen bond cleavage and the other heterolytic scission. In the heterolytic pathway a substrate radical is produced via a ferry1 (Fe = 0) species whereas in the homolytic pathway a hydroxyl radical is generated. Spin-trap - substrate radical adducts visualized in the ESR are good evidence that it is the substrate radical and not a hydroxyl radical which is involved in the catalytic cycle. The spin-traps 5,5-dimethylpyrroline-l-oxide (DMPO) and a-(4-pyridyl-l-oxide)-N-tertbutylnitrone (POBN) have been found to form adducts with alkyl substrate radicals (‘CH3, CH2OH etc). The spinadducts were identified by their characteristic hyperfine splitting constants, AN and AH, in the ESR. In none of the experiments was an oxygen based radical trapped. Thus, ‘OH (and probably ‘OOH and Oe2) can be ruled out as a participant in the s-MM0 catalysis of alkane hydroxylation.
l
These data are direct evidence for the involvement of alkyl substrate radicals in the M. capsulatus (Bath) catalysed insertion of oxygen into a C-H bond.
547
NO24
CHEMISTRY OF THE DIIRON SITE OF THE SOLUBLE MM0 HYDROXYLASE
P.C. Wilkins,a M. Fontecave,b N. Deighton,c I.D. Podmore,c M.C.R. Symons,c and H. Dalton.a aDepartment of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK bLEDSS, Universiti Joseph Fourier, 38041, Grenoble, France. ‘Department of Chemistry, University of Leicester, Leicester, LEl 7RH, UK Soluble methane monooxygenase, s-MMO, is a three component enzyme system consisting of an hydroxylase, reductase and a small regulatory protein (B). The hydroxylase contains an Fe-O-Fe center which is believed to be the site of enzyme activity. ESR spectroscopy has proved to be a very powerful method in the study of soluble methane monooxygenase from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. There have been reports of from 2 to 7.6 iron atoms per mol of hydroxylase. Titration of apo hydroxylase with metal ions, monitored by ESR, indicates that only two Mn+ are taken up per mol of hydroxylase and any excess metal ion remains free in solution. A mechanism similar to that for cytochrome P-450 has been proposed for the s-MM0 catalysed insertion of oxygen into a C-H bond. Two pathways were suggested - one involving homolytic dioxygen bond cleavage and the other heterolytic scission. In the heterolytic pathway a substrate radical is produced via a ferry1 (Fe = 0) species whereas in the homolytic pathway a hydroxyl radical is generated. Spin-trap - substrate radical adducts visualized in the ESR are good evidence that it is the substrate radical and not a hydroxyl radical which is involved in the catalytic cycle. The spin-traps 5,5-dimethylpyrroline-l-oxide (DMPO) and a-(4-pyridyl-l-oxide)-N-tertbutylnitrone (POBN) have been found to form adducts with alkyl substrate radicals (‘CH3, CH2OH etc). The spinadducts were identified by their characteristic hyperfine splitting constants, AN and AH, in the ESR. In none of the experiments was an oxygen based radical trapped. Thus, ‘OH (and probably ‘OOH and Oe2) can be ruled out as a participant in the s-MM0 catalysis of alkane hydroxylation.
l
These data are direct evidence for the involvement of alkyl substrate radicals in the M. capsulatus (Bath) catalysed insertion of oxygen into a C-H bond.
547