Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated. Resonance Raman studies show that the presence of copper in CO-bound Cu(B)Mb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu(+); it subsequently transfers from Cu(+) to Fe(2+) in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu(+) and the slow intramolecular CO transfer rate between Cu(+) and Fe(2+) in the Cu(B)Mb/Cu(+) complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound Cu(B)Mb/Cu(+) complex, the photolyzed ligand transiently binds to Cu(+) and subsequently rebinds to Fe(2+), accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the Cu(B)Mb/Cu(+) complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.

Figure 1

Structure of the binuclear center of bovine cytochrome c oxidase. The Fe–Cu_B distance is 5 Å. Cu_B is coordinated by H291, H290, and H240. H240 forms a covalent bond with Y244 due to posttranslational modification. The PDB code is 2OCC.

Figure 2

Optical absorption spectra of the deoxy and CO-bound derivatives of Cu_BMb (a) and the CO titration curves of Cu_BMb (b), in the presence and absence of 64 μM Cu⁺. The solid lines in (b) are the best-fit curves based on a single CO binding site in Cu_BMb. The K_d of CO was determined to be 420 and 360 μM for the Cu⁺-free and Cu⁺-bound Cu_BMb, respectively. In both cases, full saturation was not achieved even at a CO concentration of 1 mM. The protein concentration was 3.1 μM.

Figure 3

Resonance Raman spectra of CO-bound Cu_BMb in the presence and in the absence of 100 μM Cu⁺ (a) and the ν_Fe-CO versus ν_C-O inverse correlation plot of the data (b). The top two spectra in (a) were obtained from Cu⁺-free Cu_BMb; the bottom spectrum was obtained from Cu⁺-bound Cu_BMb, in the presence of either ¹²C¹⁶O or ¹³C¹⁸O. The ¹²C¹⁶O - ¹³C¹⁸O difference spectrum was obtained from the Cu⁺-free sample. In the difference spectrum all the heme modes cancel out, while the remaining positive and negative bands are associated with ¹²C¹⁶O- and ¹³C¹⁸O-related modes, respectively. The data shown in the ν_Fe-CO versus ν_C-O inverse correlation plot in (b) are taken either from this work or from refs , , and .

Figure 4

Transient absorption difference spectrum (t = 0.5 μs) (a) and the kinetic traces (b) obtained following photodissociation of CO from Cu_BMb. The protein and CO concentrations were 5.2 μM and 1 mM, respectively. The spectrum in (a) was obtained in the absence of Cu⁺; the solid spectrum represents the difference spectrum calculated from the equilibrium deoxy and CO-bound spectra shown in Figure 2a. The kinetic traces in (b) were obtained in the presence and in the absence of 64 μM Cu⁺, as indicated; the solid curves are the single-exponential fits of the data. In the presence of Cu, the observed rate constant for CO rebinding is 0.19 s⁻¹. The inset in (b) shows the plot of the observed rate constant of the Cu⁺-free Cu_BMb as a function of CO concentration; the k_on and k_off values were determined from the slope and intercept of the best-fit line (solid line) to be 4.8 × 102 M⁻¹ s⁻¹ and 0.18 s⁻¹, respectively.

Figure 5

CO binding kinetic traces of Cu_BMb in the presence of Cu⁺ (a) and plots of the rate constant (b) and amplitude (c) of the fast kinetic phase as a function of the CO concentration. The biexponential kinetic traces in (a) were obtained by following the changes in the deoxy band (435 nm) and in the CO-bound band (420 nm) by mixing 4 μM Cu_BMb with 50 μM CO in the presence of 100 μM Cu⁺ (final concentrations) in pH 7.4 buffer in a stopped-flow system. The rate constants in (b) were obtained from a single-exponential fit of the fast phase of the kinetic traces. The amplitudes in (c) were calculated as ΔOD_{435 nm} – ΔOD_{420 nm} of the fast phase of the kinetic traces.

Figure 6

Optical absorption (a) and resonance Raman (b) spectra of the NO adducts of ferrous Cu_BMb in the presence or absence Cu⁺. The spectra in (a) and (b) were obtained in the presence or absence of 79 and 100 μM Cu⁺, respectively. In (a), the spectrum of the Cu⁺-free deoxy Cu_BMb is shown as a reference. In (b), the spectrum of the NO-bound wild-type swMb is shown as a reference. The protein concentrations in (a) and (b) were 10 and 40 μM, respectively, prepared by adding NO to dithionite-reduced protein under anaerobic conditions.

Figure 7

Kinetic traces obtained following photodissociation of NO from Cu_BMb in the absence (a) or presence (b) of 65 μM Cu⁺. The protein and NO concentrations were 10 μM and 1.7 mM, respectively. The reduced protein solution was prepared with 9.0 mM ascorbate. The solid curves are the single-exponential fits of the data. The insets show plots of the observed rate constants as a function of NO concentration. In the absence of Cu⁺, the bimolecular rate constant was determined to be 1.6 × 10⁵ M⁻¹ s⁻¹ on the basis of the slope of the best-fit solid line. In the presence of Cu⁺, the rate (7.4 × 10⁷ s⁻¹) was independent of the NO concentration.