Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. Mössbauer and EPR studies.
Mössbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The Mössbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the Mössbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.
Fox
B G
BG
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
Surerus
K K
KK
Münck
E
E
Lipscomb
J D
JD
D004578:Electron Spin Resonance Spectroscopy; D019605:Euryarchaeota / Q000201:enzymology; D007501:Iron / Q000032:analysis; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D013057:Spectrum Analysis
D007501:Iron; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C021555:methane monooxygenase