INVESTIGATION OF THE DINUCLEAR FE CENTER OF METHANE MONOOXYGENASE BY ADVANCED PARAMAGNETIC-RESONANCE TECHNIQUES - ON THE GEOMETRY OF DMSO BINDING

We report an extensive advanced paramagnetic resonance characterizatio n of the mixed-valence dinuclear Fe center of methane monooxygenase hy droxylase (MMOH(mv)) from Methylococcus capsulatus (Mc) (Bath) and of binding to it by the exogenous ligand DMSO. We employ continuous wave and pulsed electron nuclear double resonance (ENDOR) spectroscopy, bot h at Q-band microwave frequencies, to examine N-14,N-15, H-1,H-2, C-13 , and Fe-57 nuclei. Preliminary H-1 ENDOR results were communicated pr eviously (DeRose, V. J.; Liu, K. E.; Hoffman, B. M.; Lippard, S.J. J. Am. Chem. Soc. 1993, 115, 6440-6441). ENDOR-derived N-14,N-15 hyperfin e tensors are interpreted in terms of the spin distribution on histidy l ligands bound to the dinuclear center. Determination of the Fe-57 hy perfine tensors gives a complete picture of the spin-coupled Fe2+ and Fe3+ ions. The H-1,H-2 ENDOR results disclose the presence of a bridgi ng hydroxide and an aqua ligand in both native and DMSO-treated enzyme . A novel procedure for describing the H-1 hyperfine tensor of the bri dge gives the orientation of the g-tensor relative to the cluster fram ework in both enzyme forms, information that is normally obtained only from full single-crystal EPR studies. DMSO is found to cause small pe rturbations of both histidyl ligands, and little change in the Fe-57 h yperfine tensors. However, Q-band pulsed H-2 and C-13 ENDOR measuremen ts of labeled DMSO show that this exogenous ligand binds in a distinct site with a well-ordered structure, and further indicate that it is O -bound to the Fe3+ ion of the mixed-valence cluster. The analysis, cou pled with H-2 X-band electron spin-echo envelope modulation data, plac es limitations on the possible orientation of the bound DMSO. These ge ometric restrictions have been used to guide molecular modeling of DMS O bound to the MMOH(mv) diiron active site. The results reported here provide a basis with which to study other dinuclear Fe-carboxylate pro teins.