Identification of charge-transfer transitions in the optical spectrum of low-spin ferric cytochrome P-450 Bacillus megaterium.
The optical, low temperature magnetic circular dichroism (MCD) and EPR spectra of low-spin Fe(III) cytochrome P-450 BM-3 from Bacillus megaterium, and its imidazole adduct have been measured. The MCD spectra locate new transitions over 600-700 nm and 800-1300 nm. The latter are assigned to porphyrin (a1u)-Fe(III) (dyz) charge-transfer (CT) transitions. In the case of the imidazole adduct the energy of this transition fits well to the theoretical model of Gadsby and Thomson [Gadsby, P. M. A. & Thomson, A. J. (1990) J. Amer. Chem. Soc 112, 5003-5011]. For the native enzyme, the energy of the CT band suggests co-ordination by a strongly H-bonded water ligand and the axial thiolate form of cysteine. Two transitions between 600-700 nm are detected in both derivatives. A theoretical analysis and fit of the MCD magnetisation properties shows that these transitions are polarised XY and XZ, respectively. They are assigned as thiolate sulphur py-d-shell and pz-d-shell CT transitions, analogous to the well known 695 nm band of methionine-histidine co-ordinated haem as in cytochrome c. They should prove usefully diagnostic of cysteine/Fe(III) conformational changes or protonation.
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, England.
J
J
McKnight
M R
MR
Cheesman
A J
AJ
Thomson
J S
JS
Miles
A W
AW
Munro
D001410:Bacillus megaterium / Q000201:enzymology*; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism; D008024:Ligands; D011166:Porphyrins / Q000378:metabolism; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
D008024:Ligands; D011166:Porphyrins; D003577:Cytochrome P-450 Enzyme System; D007501:Iron