Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Background: The soluble methane monooxygenase (sMMO) system in methanotroph ic bacteria uses three protein components to catalyze the selective oxidati on of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through t he sMMO reductase. Although the X-ray structure of the hydroxylase is known , little structural information is available regarding protein B. Results: Wild-type protein B from Methylococcus capsulatus (Bath) is very s usceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13- ->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mut ant forms of protein B using size exclusion chromatography and circular dic hroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) i s responsible for the proteolytic sensitivity and unusual mobility of the p rotein. We used the stable triple glycine protein B mutant to generate an a ffinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydro phobic contacts. Conclusions: A structural model is presented for protein B that explains bo th its proclivity for degradation and its anomalous behavior during size ex clusion chromatography, The model is consistent with previously published b iophysical data, including the NMR structure of the phenol hydroxylase regu latory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed reco gnition sites for the hydroxylase.