LIGAND-FIELD CIRCULAR-DICHROISM AND MAGNETIC CIRCULAR-DICHROISM STUDIES OF COMPONENT-B AND SUBSTRATE-BINDING TO THE HYDROXYLASE COMPONENT OF METHANE MONOOXYGENASE

The soluble methane monooxygenase system (MMO), consisting of a hydrox ylase (MMOH), a reductase, and component B (MMOB), catalyzes the NADH and O-2 dependent hydroxylation of methane and many other hydrocarbons . The binuclear non-heme ferrous active site cluster of the hydroxylas e-component B (MMOH-MMOB) complex in the presence of substrate and sma ll molecules has been studied using circular dichroism (CD) and magnet ic circular dichroism (MCD) spectroscopies. CD studies reveal that add ition of the alternative substrate, trans-1,2-dichloroethylene, or inh ibitor, tetrachloroethylene, induces a conformational change in the ac tive site pocket only in the presence of MMOB. Complementary MCD data indicate that this conformational change does not result in a direct c hange in the ligation of the iron atoms. Comparison of the CD/MCD data with the crystal structure of MMOH allows a tentative correlation bet ween the perturbations observed and the iron atoms affected. The bindi ng of MMOB to MMOH distorts the ligand field environment of one iron, while substrate binding in the presence of MMOB perturbs the other, th erefore providing insight into the regulatory role of MMOB and into th e participation of the two iron centers in the reaction. Anion binding to the MMOH-MMOB complex was also investigated. No spectral perturbat ion by small molecules (K-B < 30 M(-1)) in reduced MMOH and the MMOH-M MOB complex was observed, suggesting that the hydroxylase active site pocket is less electrophilic than other binuclear non-heme iron protei ns, consistent with its role in charge donation to activate dioxygen.