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

The interaction between the hydroxylase component of methane monooxygenase (MMO) from Meth~ococcus capsu/atus (Bath), the coupling protein of the MMO enqme system (component B) and substrate has been investigated by using Fe K~dge x-ray absorption spectros-copy (MS). Fe K-edge extended x-ray abso~tion fine stmcture (EXAFS) studies of the semimet form of the hydroxylase in the presence of the coupling protein, 1-brom@l-propene, and both the coupling protein and 1-bromol-propene revealed small differences in the appearance of the EXAFS above k= 8 A-' ascompared to the non-complexed 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 2.06-2.08 A, which is comparable to the distance found for the non-complexed form, 2.06-2.09A. 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 con-sistent'decrease in the Fe-Fe distance of the B-complexed hydroxylase samples, from 3.42 A to 3.39 A. When only 1-bromo-l-propene was present the distance remained unchanged. Similafiy, differences were seen in the EXAFS of the reduced forms of the hydroxylase complex above k= 8.5 A-', but the average Fecoordination as determined by fits to the data was similar to that of the non-complexed reduced hydroxylase. For the complexed forms of the reduced hydroxylase, an average fimt shell Fe-O/N distance of 2.11-2.14A was found, compa-— rable to the 2.15 A distance found for the non-complexed reduced hydroxylase, but a change in the distribution of long vs. shod distance contributions was again observed when component B was p;esent. High resolution Fe K-edge edge spectra of the Bcomplexed samples revealed a shoulder on the-rising edge of the semimet fom of the hydroxylase, suggesting a change in covalency at the Fe site. Fufihenore, 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 fomation, resulting in a distortion in coordination , a change in the covalency of the Fe center, andor a change in the ligation of the Fe center. Additionally, comparison of EXAFS results for …