Molecular and spectroscopic analysis of the cytochrome cbb(3) oxidase from Pseudomonas stutzeri.
Cytochrome cbb(3) oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb(3) oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UV-visible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.
Pitcher
Robert S
RS
Centre for Metalloprotein Spectroscopy and Biology and the School of Biological Sciences, University of East Anglia, Norfolk NR4 7TJ, United Kingdom.
Cheesman
Myles R
MR
Watmough
Nicholas J
NJ
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared
C414443:cbb3 oxidase; D003576:Electron Transport Complex IV