Figure 1.
Formation of the CO compound of isolated cytochrome bd from E. coli in MV state.
Shown is static difference absorbance spectrum of CO-treated enzyme versus a spectrum of as-isolated enzyme. The experiments were performed at 20 °C in buffer containing 50 mM Hepes, 50 mM Ches, 0.1 mM EDTA, and 0.05% sodium N-lauryl-sarcosinate (pH 8.0) in an optical cell of 10 mm pathway. Concentrations of enzyme and CO were 1.9 µM and 1 mM respectively.
Figure 2.
Absorbance changes accompanying photodissociation and subsequent recombination of CO with cytochrome bd in MV state.
Kinetics at selected wavelengths. The kinetic data points (noisy traces) are shown with their best fits (smooth lines). Buffer: 50 mM Hepes, 50 mM Ches, 0.1 mM EDTA, 0.05% sodium N-lauryl-sarcosinate, pH 8.0. Enzyme, 1.9 µM; CO, 1 mM. Optical pathway, 10 mm; excitation, 532 nm; temperature, 20 °C.
Figure 3.
Absorbance changes following flash photolysis of the CO complex with cytochrome bd in MV state.
(a) Transient spectra in the Soret and visible regions at a delay time of ∼1.5 µs (versus pre-trigger). Dark cyan dashed lines: model spectra of electron transfer from heme d to heme b595 constructed from individual reduced-minus-oxidized difference absorption spectra of the hemes published in [56]. Red solid line in right panel: expected spectrum. The expected spectrum is the transient spectrum for the R enzyme at a delay time of ∼1.5 µs (taken from Fig. 5a, right panel) reduced by a factor of 2.5 to account for geminate recombination of CO to heme d that occurs in the MV enzyme at early times [41], [43], [46]. (b), (c) and (d) Difference spectra of kinetic phases with τ ∼ 16 µs, 180 µs and 30 ms respectively. Buffer: 50 mM Hepes, 50 mM Ches, 0.1 mM EDTA, 0.05% sodium N-lauryl-sarcosinate, pH 8.0. Enzyme, 1.9 µM; CO, 1 mM. Optical pathway, 10 mm; excitation, 532 nm; temperature, 20 °C.
Figure 4.
Absorbance changes accompanying photodissociation and subsequent recombination of CO with cytochrome bd in R state.
Kinetics at selected wavelengths. The kinetic data points (noisy traces) are shown with their best fits (smooth lines). Buffer: 50 mM Hepes, 50 mM Ches, 0.1 mM EDTA, 0.05% sodium N-lauryl-sarcosinate, pH 8.0. Enzyme, 1.9 µM; CO, 1 mM. Optical pathway, 10 mm; excitation, 532 nm; temperature, 20 °C.
Figure 5.
Absorbance changes following flash photolysis of the CO complex with cytochrome bd in R state.
(a) Transient spectra in the Soret and visible regions at a delay time of ∼1.5 µs (versus pre-trigger). (b), (c) and (d) – Difference spectra of 20 µs, combined 0.2–3 ms and 24 ms kinetic phases respectively. Buffer: 50 mM Hepes, 50 mM Ches, 0.1 mM EDTA, 0.05% sodium N-lauryl-sarcosinate, pH 8.0. Enzyme, 1.9 µM; CO, 1 mM. Optical pathway, 10 mm; excitation, 532 nm; temperature, 20 °C.
Figure 6.
Proposed scheme for backflow of electrons in cytochrome bd in MV state.
Each rhombus represents one of the three hemes in cytochrome bd. Filled or semi-filled rhombus denotes that site is in the reduced state. Empty rhombus denotes that site is in the oxidized state. During the backflow reaction (observed in ∼4% of the enzyme at 1 mM CO) the electron from heme d transfers sequentially to heme b595 (A→B transition, τ<1.5 µs) and heme b558 (B→C transition, τ∼16 µs). Finally, the electron equilibrates between the redox sites with respect to their redox potentials. A→D and C→D transitions describe recombination of CO to 96% and 4% of heme d, respectively.
Figure 7.
Proposed intermediate states of the E. coli cytochrome bd during photolysis of CO and its subsequent recombination.
The minimal scheme shows the porphyrin plane of heme d, the central iron atom, changes in heme ligation and the time constants for the resolved transitions of heme d. MV (a) and R (b) are the redox states of the enzyme. In the MV state of the enzyme, photodissociation of CO from heme d is accompanied by binding of L at the opposite side of the heme. CO recombines with heme d with τ∼16 µs yielding a transient hexacoordinate state (CO-Fe2+-L). Then L is dissociated from heme d with τ∼30 ms. In the R state of the enzyme, L is a permanent undissociable proximal ligand to heme d. In this case, there are two transitions with regard to heme d: after photolysis CO leaves the heme pocket and then returns to heme d with τ∼20 µs.