An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center.
Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.
Davydov
R
R
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
Valentine
A M
AM
Komar-Panicucci
S
S
Hoffman
B M
BM
Lippard
S J
SJ
D001665:Binding Sites; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D005615:Freezing; D008772:Methylococcaceae / Q000201:enzymology*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology; D012995:Solubility
D005290:Ferric Compounds; D005296:Ferrous Compounds; D010636:Phenols; D010105:Oxygenases; C021555:methane monooxygenase; D004121:Dimethyl Sulfoxide