X-ray Absorption Spectroscopic Studies of the Diiron Center in Methane Monooxygenase in the Presence of Substrate and the Coupling Protein of the Enzyme System

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-l-propene, and both the coupling protein and 1-bromo-l-propene revealed small differences in the appearance of the EXAFS above k = 8 Å⁻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 Å, which is comparable to the distance found for the noncomplexed form, 2.06–2.09 Å. 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 Å. When only 1-bromo-l-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 Å⁻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 Å was found, comparable to the 2.15 Å 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. High resolution Fe K-edge edge spectra of the B-complexed samples revealed a shoulder on the rising edge of the semimet form of the hydroxylase, suggesting a change in covalency at the Fe site. Furthermore, differences in the edge spectra of the reduced forms of the hydroxylase suggested that the coupling protein and substrate influence the electronic environment of the Fe center. Together, these results show that a subtle change in the Fe environment of the hydroxylase occurs upon complex formation, resulting in a distortion in coordination, a change in the covalency of the Fe center, and/or a change in the ligation of the Fe center. Additionally, comparison of EXAFS results for a brominated model compound to that for 1-bromo-l-propene substrate complexed with hydroxylase provided no evidence that the bromine atom of bound substrate is located within about 3.5 Å of an Fe atom. This result makes it unlikely that olefins form a π complex to Fe in the MMO hydroxylase.