X-ray Absorption Spectroscopic Studies of the Dinuclear Iron Center in Methane Monooxygenase and the Sulfure and Chlorine Centers in Photographic Materials*

The Diiron Center in Methane Monooxygenase. The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus (Bath) and Methylosinus trichosporium (OB3b) has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(III)Fe(III) oxidized state of the hydroxylase from M. capsuZatus (Bath) consists of approximately 6 N and 0 atoms at an average distance of 2.04 A. The Fe-Fe distance was determined to be 3.4 A. No-evidence for the presence of a short 0x0 bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MM0 is significantly different from the active sites of the dinuclear iron proteins hemerythrin and ribonucleotide reductase. In addition, the results’of the first shell fits suggest that there are more oxygen than nitrogen donor ligands. The active sites of the photoreduced Fe(III)Fe(II) semimet form of the hydroxylase from both M. capsdam (Bath) and M. trichosporium (OB3b) consist of approximately 6 N and 0 atoms at an average distance of 2.06 2.09 A with an Fe interaction at 3.41 3.43 A. This implies that the diiron center of the hydroxylase from the two species are structurally similar. In addition, the results of the second shell fits to the hydroxylase suggest that there is a shell of low-2 atoms at 3.0 8, in both the diferric and semimet active sites. Upon reduction to the Fe(II)Fe(II) reduced form, the average first shell distance increased to 2.15 %, and the Fe-Fe interaction was no longer detected. The Fe K-edge EXAFS showed only minor metrical changes in the coordination environment of the hydroxylase iron center due to the presence of substrate and component B, the regulatory protein of the MM0 enzyme system. This finding was true for the complexes of semimetand reduced hydroxylase. The changes seen occured in the first coordination sphere. In particular, the presence of component B seemed to have an effect on the distance distribution of first shell atoms. No evidence of a Br contribution was seen in the EXAFS of the hydroxylase in the presence of a brominated substrate. This suggests that the site of interaction between the hydroxylase and substrate is more than 4 A from the iron center. The presence of substrate and component B was found to modify the Fe Kedge spectra of the hydroxylase. The change seen in the spectra of the semimet samples is consistent with an increase in the covalency of the iron center. The appearance of the edges of the reduced forms of the hydroxylase suggest that the presence of substrate or component B inhibits the reduction of the diferric hydroxylase to a diferrous state. These studies suggest that the changes seen in the hydroxylase diiron site in the presence of