Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath).
Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol. The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function. In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli. Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure. Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins. The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR. The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (micro-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes. Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD.
Merkx
Maarten
M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Lippard
Stephen J
SJ
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D010105:Oxygenases; C021555:methane monooxygenase