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 fi...