Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy

Abstract

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.

Transmutation of a heme protein.

“…Second, the intense band at 746 cm-' is a characteristic feature of resonance Raman spectra of chlorins (26). Finally, the unusual resonance Raman bands around 1240 cm-' are similar to features recently reported for the spectrum of cytochrome d (27), which is known to possess a chlorin prosthetic group. greater oxidizing potential ofN57C-C suggests that additional modification of the prosthetic group has resulted in further substitution on the macrocycle.…”

Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site

“…Both phases probably reflect the conversion of A to F in different subpopulations of the enzyme. The conclusion that the final product of this reaction is the F state was confirmed by the absolute absorption spectrum taken after completion of the reaction (2 s) and showing a peak at 680 nm, diagnostic of the heme d oxoferryl species (42). Recombination of CO with heme d after photolysis of the mixed-valence cytochrome bd includes (i) CO rebinding to the heme d after the flash and (ii) the return of the electrons from the transiently reduced hemes b 558 and b 595 to heme d (11).…”

Cyanide-binding Site of bd-type Ubiquinol Oxidase from Escherichia coli

“…the space between the two metal centers) must be highly restricted, and the ligand-binding pocket itself would be very hydrophobic. The hydrophobic nature of the ligand-binding pocket is consistent with the unusual stability of the oxygenated species as well as the oxoferryl (Fe 4ϩ ϭO) adduct (10,11). This might be one of the reasons why bd-type ubiquinol oxidase is more resistant to anionic inhibitors such as cyanide and azide than bo-type ubiquinol oxidase (2,3).…”

“…The dioxygen molecule forms a very stable adduct with ferrous cytochrome d showing its Fe 2ϩ -O 2 stretching vibration at 568 cm Ϫ1 (essentially identical to that of myoglobin) (10), and thus the enzyme in the air-oxidized state is actually an oxygenated form. The cytochrome d moiety also forms a remarkably stable oxoferryl (Fe 4ϩ ϭO) adduct (11). Subunit I contains cytochrome b 558 that shows its ␣ and ␤ peaks at 562 and 532 nm, respectively, in the reduced state (12) and most likely forms the ubiquinol-8 binding site (13).…”