X-ray crystal structures of the hydroxylase component (MMOH) of soluble methane monooxygenase (MMO) show that it contains a dihydroxo-bridged diiron cluster. The complete enzyme system also includes a reductase (MMOR) and a B (MMOB) component. The reductase serves to facilitate transfer of 2 electrons to diferric MMOH, thereby initiating the catalytic cycle, but it also acts to shift the electrode potential values of the diiron cluster so that the two electron reduced state is favored over the unreactive mixed valence state. MMOB acts as a gating protein to accelerate the reaction of diferrous MMOH with O 2 by 1000-fold so that this reaction is not rate limiting for catalysis. These functions of MMOB and MMOR are mediated through formation of specific complexes with MMOH that result in structural changes at each oxidation state of the diiron cluster. Transient kinetic and reactivity studies have revealed that a single turnover of diferrous MMOH in the presence of O 2 , substrate, and MMOB occurs through at least 5 intermediates: compounds O, P, Q, R, and T. Q is the intermediate that oxidizes hydrocarbons, in most cases by net transfer of an oxygen atom derived from O 2 . The 2 H kinetic isotope effect for the transfer of oxygen into 2 H-labeled methane is 50-100 suggesting a discrete C-H bond breaking step to generate the putative radical intermediate, R. R is too short lived to be directly observed, but has been inferred from the observation of inversion of stereochemistry during oxidation of chiral substrates. The kinetics of formation and decay of the bright yellow Q allow it to be trapped in >60% yield for spectroscopic studies conducted in collaboration with L. Que and E. Munck. Mossbauer spectra, considered in the light of the spectra of Fe(IV) containing model complexes, show that the irons of Q reside in nearly identical environments and that they are both in the high spin Fe(IV) oxidation state. EXAFS studies show that the diiron cluster of Q has a short Fe-Fe distance of 2.46 9 and one short Fe-O bond (per Fe) of 1.77 9. Based on the model compounds these distances are only compatible with a diamond core structure in which the irons are bridged by two single atoms. Each bridge has one short and one long bond showing that each iron is more strongly associated with one bridging oxygen that the other. Unsubstituted oxo-bridges in Q arranged as a head to tail Fe(IV)=O dimer would account for the EXAFS, Mossbauer, and visible spectroscopic features. The basis for the unique reactivity of this structure is being explored through kinetic, spectroscopic, and chemical studies of Q and parallel studies of high valent model compounds. The roles of MMOB and MMOR in the regulation of this catalysis are being studied through structural and dynamic studies of component interaction.