Stationary and time-resolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli.
The heme environments of Met(95) and His(77) mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(CO) plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the nu(Fe-His) band but gave a nu(Fe-Im) band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a five-coordinate heme complex. Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2). Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS.
Sato
Akira
A
School of Advanced Sciences, The Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa 240-0193, Japan.
Sasakura
Yukie
Y
Sugiyama
Shunpei
S
Sagami
Ikuko
I
Shimizu
Toru
T
Mizutani
Yasuhisa
Y
Kitagawa
Teizo
T
D002248:Carbon Monoxide / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008715:Methionine / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman; D013997:Time Factors
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D008715:Methionine; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli; D010100:Oxygen