Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Abstract

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Figure 1

Absorption characteristics of cytochrome bd in the Soret band. (A) Static absorption spectra of cytochrome bd: (a) fully reduced; (b) CO complex of the fully reduced state; (c) as-prepared oxygenated state; (d) CO complex of the MV state. (B) Evolution of spectral changes induced by femtosecond excitation of the fully reduced cytochrome bd at 590 nm. The difference spectra have been recorded versus the static R state at the indicated time intervals after the photoexcitation. (C) Spectra of the subpicosecond component (0.45–0.9 ps for different states) of the photobleaching induced in various states of cytochrome bd. Experiments have been carried out in the indicated redox and ligand binding states at λ_exc indicated in brackets. Solid line overlaying spectrum b: approximation with a single Gaussian. (D) γ-Absorption band of ferrous heme b₅₉₅ resolved by photobleaching of the R state with 590-nm excitation (crosses, redrawn from C, curve b) has been approximated by a single Gaussian (solid line a).

Figure 2

Photodissociation of CO from the R (A) and MV states (B) of cytochrome bd. The absorption changes observed with the MV-CO complex have been expanded 2-fold. (C) Kinetics of the photoinduced changes in the two states. The responses have been normalized at t = 20 ps.

Figure 3

A red shift of ferrous heme b₅₉₅ absorption band induced by CO dissociation from heme d. (A) (a) Absorption changes induced by CO photodissociation from the MV and (b) static unliganded-CO − liganded spectrum of the R state (difference between curves a and b of Fig. 1A). The curves have been normalized by aligning their blue parts where they have similar line shapes. The scale bar refers to the R-CO state response. (B) Curve a gives the difference between spectra b and a in A. For comparison are shown simulated difference spectra for a 20-nm red shift corresponding to CO dissociation from heme b₅₉₅ in ≈10% of cytochrome bd (b) and a 1.2-nm red shift of the entire population of b₅₉₅ (c).

Figure 4

Evidence for an unrelaxed intermediate state of ferrous heme b₅₉₅ after CO photodissociation from heme d. (A) Absorption changes induced by CO dissociation from the R-CO state of cytochrome bd shortly after photolysis (a) and under static conditions (b). Curve a corresponds to the difference spectrum resolved by global analysis as a residual constant phase after relaxation of the excited states. Curve b is the difference spectrum (inverted) induced by addition of 20 μM CO to the fully reduced cytochrome bd (the same curve as in Fig. 3A). The spectra have been normalized by the height of the 442-to 444-nm peak. The ΔA scale bar refers to curve a. (B). The experimental difference between spectra a and b in A (●) has been simulated (the line) as narrowing of the b₅₉₅ 440-nm band by 10% plus a 17-nm blue shift of this band in 10% of cytochrome bd. The Gaussian approximation of the band in Fig. 1D has been used for modeling. (Inset) After subtracting the contribution of b₅₉₅ band narrowing, the difference between the picosecond and static CO photodissociation spectra (crosses) is dominated by the 17-nm blue shift of heme b₅₉₅ (solid curve).