Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Abstract

Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau approximately 12 micros, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau approximately 14 ns, 14 micros, and 280 micros. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-micros component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in approximately 4% of the enzyme population. The final, 280-micros component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium.

Fig. 1

Absorption changes measured after photodissociation of CO from the E. coli membranes containing the WT cytochrome bd in the R-CO state. Main panel: Kinetics of absorption changes at selected wavelengths during CO recombination. The kinetic data points (symbols) are shown with their best fits to single exponentials (solid lines) yielding τ ~ 12 µs. Arrow indicates the moment of laser flash. Inset: Transient absorption spectrum at a delay time of 5 ns. For conditions, see the Materials and methods section.

Fig. 2

Absorption changes measured after photodissociation of CO from the E. coli membranes containing the WT cytochrome bd in the MV¹-CO state. (A) Transient absorption spectra at delay times of 5 ns, 200 ns, and 60 µs. (B) Kinetics of absorption changes at selected wavelengths during CO recombination. The kinetic data points (symbols) are shown with their best fits (solid lines). The kinetics at 435 nm is fitted to the sum of three exponentials yielding τ ~ 14 ns, 14 µs, and 280 µs. 417.5 nm has been selected as the wavelength isosbestic for the 14 ns and 280 µs components, therefore for that kinetics a one-exponential fit with τ ~ 14 µs is sufficient. Arrow indicates the moment of laser flash. (C) Spectra of the absorption changes associated with the 14-ns, 14-µs, and 280-µs components. The spectrum of the 14-ns phase is calculated as the difference between the transient spectra measured at delay times of 5 ns and 200 ns. The spectrum of the 280-µs phase is the spectrum measured 60 µs after the flash. The amplitude of the latter has been divided by e^−60/280 to correct for the decay at 60 µs. The spectrum of the 14-µs phase is calculated as the difference between the transient spectrum measured at a delay time of 200 ns and the spectrum of the 280-µs phase. (D) Difference between R-CO and MV¹-CO transient spectra at a delay time of 5 ns. The spectra are normalized at 445 nm (spectrum MV¹-CO multiplied by 7.5) as the spectral properties at this wavelength are independent of the oxidation state of the b-hemes [41].

Fig. 3

Absorption changes measured after photodissociation of CO from the E. coli membranes containing the E445A mutant cytochrome bd in the MV²-CO state. (A) Transient absorption spectra at delay times of 5 ns, 200 ns, and 1.5 ms. (B) Kinetics of absorption changes at selected wavelengths during CO recombination. The kinetic data points (symbols) are shown with their reasonable fits to the sum of two exponentials (solid lines) yielding τ ~ 14 ns, and 42 µs. Approximation with three exponentials does not improve the fit significantly. Arrow indicates the moment of laser flash. (C) Difference between transient absorption spectra of the WT cytochrome bd in the R-CO state and the E445A mutant cytochrome bd in the MV²-CO state at a delay time of 5 ns (normalized at 445 nm). (D) Comparison of the spectra of the absorption changes associated with the nanosecond components for the WT cytochrome bd in the MV¹-CO state and the E445A mutant cytochrome bd in the MV²-CO state (normalized on the maximum). The latter spectrum is a difference between the MV²-CO E445A transient spectra at delay times of 5 ns and 200 ns.

Fig. 4

(A) Minimal scheme of geminate recombination phases of CO starting from the MV¹-CO and R-CO states. Two different configurations of dissociated CO in the protein (d^red……CO_i i=I, II) are required to explain the two geminate recombination phases. The ratio of forward and backward rates from these configurations is roughly estimated from the amplitudes of the phases; the sums of the rates correspond to the experimentally observed rates. The state (d_red + CO) denotes a state where CO has escaped from the protein. In this minimal scheme, if heme b₅₉₅ is reduced prior to dissociation of the heme d-CO bond, geminate recombination from the (d_red……CO_I) state does not compete efficiently with population of the (d^red……CO_II) state. (B) Minimal scheme of bimolecular CO recombination and electron transfer starting from the MV¹-CO. The first dissociation step comprises all steps in scheme (A). The ratio of forward and backward rates from the CO dissociated d_red is roughly estimated from the amplitudes of the phases; the sums of the rates correspond to the experimentally observed rates.