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 monooxygena se (MMO) from Methylococcus capsulatus (Bath), the coupling protein of the MMO enzyme system (component B), and substrate has been investiga ted by using Fe K-edge X-ray absorption spectroscopy (XAS). Fe K-edge extended X-ray absorption fine structure (EXAFS) studies of the semime t form of the hydroxylase in the presence of the coupling protein, 1-b romo-1-propene, and both the coupling protein and 1-bromo-1-propene re vealed small differences in the appearance of the EXAFS above k = 8 An gstrom(-1) as compared to the noncomplexed hydroxylase. No dramatic ch ange 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 hyd roxylase ranged between 2.06 and 2.08 Angstrom, which is comparable to the distance found for the noncomplexed form, 2.06-2.09 Angstrom. Alt hough 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 co nsistent 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 abov e 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 hy droxylase. For the complexed forms of the reduced hydroxylase, an aver age first shell Fe-O/N distance of 2.11-2.14 Angstrom Was found, compa rable to the 2.15 Angstrom distance found for the noncomplexed reduced hydroxylase, but a change in the distribution of long vs short distan ce contributions was again observed when component B was present. High resolution Fe K-edge edge spetra 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, differen ces in the edge spectra of the reduced forms of the hydroxylase sugges ted that the coupling protein and substrate influence the electronic e nvironment of the Fe center. Together, these results show that a subtl e 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 mod el compound to that for 1-bromo-1-propene substrate complexed with hyd roxylase provided no evidence that the bromine atom of bound substrate is located within about 3.5 Angstrom of an Fe atom. This result makes it unlikely that olefins form a pi complex to Fe in the MMO hydroxyla se.