Oxidation-reduction potentials of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

• Published in: Biochemistry (Easton) Vol. 33; no. 3; pp. 713 - 722
• Main Authors: Paulsen, Kim E, Liu, Yi, Fox, Brian G, Lipscomb, John D, Munck, Eckard, Stankovich, Marian T
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-01-1994
• Subjects: Alkenes - chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Electron Spin Resonance Spectroscopy; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Mechanisms. Catalysis. Electron transfer. Models; Methylococcaceae - enzymology; Methylosinus trichosporium; Models, Chemical; Molecular biophysics; Oxidation-Reduction; Oxidoreductases; Oxygenases - chemistry; Physical chemistry in biology; Potentiometry; Thermodynamics; Redox state; Iron Complexes; Methylosinus trichosporium; Enzyme; Association dissociation; Reaction mechanism; Bacteria; Methane monooxygenase; Oxidoreductases; Redox potential; Subunit; Conformational transition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00169a013

Methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB) protein components. MMOH contains two oxygen-bridged dinuclear iron clusters that are the sites of O2 activation and hydrocarbon oxidation. Each cluster can be stabilized in diferric [Fe(III).Fc(III)], mixed-valence [Fe(II).Fe(III)], and diferrous [Fe(II).Fe(II)] redox states. We have correlated the EPR spin quantitation of the S = 1/2 mixed-valence state with the system electrode potential to determine both formal redox potential values for MMOH at 4 degrees C: E1 degrees' = +76 +/- 15 mV and E2 degrees' = +21 +/- 15 mV (Em = +48 mV, 61% maximum mixed-valence state). Complementary Mössbauer studies of 57Fe-enriched MMOH allowed all three redox states to be quantitated simultaneously in individual samples and revealed that the distribution of redox states was in accord with the measured potential values. EPR spectra of partially reduced MMOH showed that the apparent midpoint potential values of MMOH-MMOR, MMOH-MMOR-MMOB, and MMOH-MMOR-MMOB-substrate complexes were slightly more positive than that of MMOH alone. In contrast, the MMOH-MMOB complex appeared to have a substantially more negative redox potential. The formal redox potential values of the latter complex were determined to be E1 degrees' = -52 +/- 15 mV and E2 degrees' = -115 +/- 15 mV, respectively, at 4 degrees C (Em = -84 mV, 65% maximum mixed-valence state). This negative 132-mV shift in the midpoint potential of MMOH coupled to MMOB binding suggests that MMOB binds approximately 10(4) more strongly to the diferric state of MMOH than to the diferrous state. Since the potential shift is strongly negative, and since a nearly constant separation between the two formal potential values of MMOH is maintained when MMOB binds, the role of the MMOB-MMOH complex must not be to thermodynamically stabilize the formation of the diferrous cluster which is the form that reacts with O2 during catalysis. However, MMOB binding may provide kinetic stabilization of the diferrous state and/or modulation of the interaction of MMOH with O2 and hydrocarbon substrates. Such roles may be effected through cyclic association and dissociation of the MMOB-MMOH complex as MMOH oscillates between redox states during catalysis, thereby dynamically altering the affinity of this complex.