Background: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation 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 the 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 susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts.
Conclusions: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.