Abstract
During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the (FeFeII)-Fe-II state (H-red). O-2 binds rapidly at a site away from the cluster to form the (FeFeII)-Fe-II intermediate O, which converts to an (FeFeIII)-Fe-III-peroxo intermediate P and finally to the (FeFeIV)-Fe-IV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of H-red and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of H-red and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mossbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mossbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the (FeFeII)-Fe-II state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the (FeFeII)-Fe-II center rather than oxidation. The similarity of the optical and Mossbauer spectra of H-red, O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O-2 to be activated for hydrocarbon oxidation.
