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

Abstract

The heme environments of Met95 and His77 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 (νFe-His) at 214 cm-1, and the νFe-Co versus νCo 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 νFe-His band but gave a νFe-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 His77 and Met95, and that Met95 is displaced by CO and O2. Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met95 following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS.