GATING EFFECTS OF COMPONENT-B ON OXYGEN ACTIVATION BY THE METHANE MONOOXYGENASE HYDROXYLASE COMPONENT

Component B (MMOB) of the soluble methane mono oxygenase (MMO) system accelerates the initial velocity of methane oxidation by up to 150-fold by an unknown mechanism. The active site of MMO contains a diferric, hydroxo-bridged diiron cluster located on the hydroxylase component (MMOH). This cluster is reduced by the NAD(P)H-coupled reductase component to the diferrous state, which then reacts with O-2 to yield two reaction cycle intermediates sequentially termed compounds P and Q. The rate of compound P formation is shown here to be independent of O-2 concentration, suggesting that an MMOH-O-2 complex (compound O) is (similar to irreversibly) formed before compound P. Compound Q is capable of reacting with hydrocarbons to yield the MMOH-product complex, compound T. It is shown here that MMOB accelerates catalysis by increasing similar to 1000-fold the rate of O-2 association and reaction with diferrous MMOH leading to compound P. Modeling of the single turnover reaction in the presence of substoichiometric MMOB suggests that MMOB also accelerates the compound P to Q conversion by similar to 40-fold. Due to this O-2-gating effect of MMOB, either compound Q or T becomes the dominant species during turnover, depending upon the substrate concentration and type. Because these are the species that either react with substrate (Q) or release product (T), their buildup maximizes the turnover rate. This is the first direct role in catalysis to be recognized for MMOB and represents a novel method for oxygenase regulation.