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 hydroxylase (MMOH), a reductase, and component B (MMOB), catalyzes the NADH and O₂ dependent hydroxylation of methane and many other hydrocarbons. The binuclear non-heme ferrous active site cluster of the hydroxylase-component B(MMOH-MMOB) complex in the presence of substrate and small molecules has been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. CD studies reveal that addition of the alternative substrate, trans-1,2-dichloroethylene, or inhibitor, tetrachloroethylene, induces a conformational change in the active site pocket only in the presence of MMOB. Complementary MCD data indicate that this conformational change does not result in a direct change in the ligation of the iron atoms. Comparison of the CD/MCD data with the crystal structure of MMOH allows a tentative correlation between the perturbations observed and the iron atoms affected. The binding of MMOB to MMOH distorts the ligand field environment of one iron, while substrate binding in the presence of MMOB perturbs the other, therefore providing insight into the regulatory role of MMOB and into the participation of the two iron centers in the reaction. Anion binding to the MMOH-MMOB complex was also investigated. No spectral perturbation by small molecules (K(B) < 30 M^-1) in reduced MMOH and the MMOH-MMOB complex was observed, suggesting that the hydroxylase active site pocket is less electrophilic than other binuclear non-heme iron proteins, consistent with its role in charge donation to activate dioxygen.