The biosynthesis and assembly of protein A of soluble methane monooxygenase of Methylococcus capsulatus (Bath).
Protein A of soluble methane monooxygenase (EC 1.14.13.25) of Methylococcus capsulatus (Bath) is the hydroxylase component of the enzyme complex, capable of inserting an atom of oxygen into methane. The protein possesses an unusual non-heme iron center consisting of two mu-hydroxo-bridged antiferromagnetically coupled iron atoms. It was possible to remove the iron center of protein A by subjecting the protein to freeze/thaw cycles or by dialysis against 8-hydroxyquinoline. Incubation of iron-depleted protein A with iron-EDTA and dithiothreitol resulted in the reassembly of the iron center of protein A as judged by restoration of enzyme activity (typically approximately 3 times the original activity) and by ESR spectroscopic methods. Reconstitution was inhibited by a number of metal ions, but none of the metals tested proved capable of replacing iron in the oxidation of methane. A number of iron-chelating agents exhibited weak inhibition of reconstitution. The KM(app) for iron in the reconstitution reaction was 133 microM. In vitro translation of total RNA isolated from M. capsulatus (Bath) produced protein A subunits equal to the native molecular weight, suggesting that there is no precursor form of this protein. Addition of the in vitro translated protein A to the reconstitution system resulted in low levels of hydroxylase activity suggesting that the subunits of protein A are self-assembling, a supposition supported by the presence of only one protein band on nondenaturing gels. Taken together, these data suggest that the subunits of protein A are synthesized in their native, mature form and self-assemble into an apoprotein A, into which the iron center is inserted.
Green
J
J
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Dalton
H
H
D002413:Cations, Divalent; D004578:Electron Spin Resonance Spectroscopy; D019605:Euryarchaeota / Q000201:enzymology*; D007501:Iron / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D010105:Oxygenases / Q000096:biosynthesis* / Q000378:metabolism
D002413:Cations, Divalent; D046911:Macromolecular Substances; D007501:Iron; D010105:Oxygenases; C021555:methane monooxygenase