The interaction among the hydroxylase component of methane monooxygenase (MMO) from Methylococcus capsulatus (Bath), the coupling protein of the MMO enzyme system (component B), and substrate has been investigated by using Fe K-edge X-ray absorption spectroscopy (XAS). Fe K-edge extended X-ray absorption fine structure (EXAFS) studies of the semimet form of the hydroxylase in the presence of the coupling protein, 1-bromo-1-propene, and both the coupling protein and 1-bromo-1-propene revealed small differences in the appearance of the EXAFS above k = 8 Angstrom(-1) as compared to the noncomplexed hydroxylase. No dramatic change in the Fe coordination was seen in fits to the data. The average first shell Fe-O/N distance for the complexed forms of the semimet hydroxylase ranged between 2.06 and 2.08 Angstrom, which is comparable to the distance found for the noncomplexed form, 2.06-2.09 Angstrom. Although the average first shell coordination was similar for all samples, a difference was seen in the distribution of long vs short distance contributions to the first shell coordination sphere for samples with component B present. This difference was accompanied by a small but consistent decrease in the Fe-Fe distance of the B-complexed hydroxylase samples, from 3.42 to 3.39 Angstrom. When only 1-bromo-1-propene was present, the distance remained unchanged. Similarly, differences were seen in the EXAFS of the reduced forms of the hydroxylase complex above k = 8.5 Angstrom(-1), but the average Fe coordination as determined by fits to the data was similar to that of the noncomplexed reduced hydroxylase. For the complexed forms of the reduced hydroxylase, an average first shell Fe-O/N distance of 2.11-2.14 Angstrom Was found, comparable to the 2.15 Angstrom distance found for the noncomplexed reduced hydroxylase, but a change in the distribution of long vs short distance contributions was again observed when component B was present.