Ligand trapping by cytochrome c oxidase: implications for gating at the catalytic center

Abstract

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe(a3)(3+)-Cu(B)(2+)) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe(a3)(3+)-CN-Cu(B)(2+) complex. Instead the cyanide-induced transition of a preformed Fe(a3)(3+)-N(3)-Cu(B)(2+) to the ternary complex of Fe(a3)(3+)-N(3) Cu(B)(2+)-CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu(B) blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu(B) and that, during catalysis, ligands present at this copper center control access to the iron of heme a(3) from the bulk medium.

FIGURE 1.

Influence of azide on the optical spectrum of oxidized cytochrome oxidase and the spectral change triggered by the addition of cyanide. A, the Soret band spectra of oxidized CcO (O), in the presence of 0.2 mm NaN₃ (O.N₃) and 2 mm NaCN (O.CN). B, the kinetics of spectral change in the Soret band initiated by the addition of 2 mm NaCN to oxidized CcO (O) and to the solution of enzyme containing 0.2 mm NaN₃ (+N₃). C, the dependence of the initial rates of the spectral change induced by 2 mm NaCN on the concentration of azide in the solution. The dotted guideline is used to highlight two opposite effects of azide. Conditions of measurements are as follows: concentration of CcO, 4.2 μm; buffer, 100 mm Hepes, pH 7.8, 50 mm K₂SO₄, 0.05% DM; temperature, 23 °C.

FIGURE 2.

FTIR spectra of free azide and in complex with cytochrome oxidase. pH 4.1, the spectrum of 1 m NaN₃ in water at pH 4.1. The spectrum has been displaced along the vertical axis. pH 7.8, the spectrum of CcO with one equivalent of NaN₃ (800 μm CcO + 800 μm NaN₃) at pH 7.8.

FIGURE 3.

FTIR spectra of cytochrome oxidase complexed with cyanide. CcO+CN, the spectrum of 700 μm CcO with 1 mm NaCN in a Hepes buffer at pH 7.8. The spectrum has been displaced along the vertical axis. filtration, the spectrum of the complex of CcO with cyanide after gel filtration.

FIGURE 4.

FTIR spectra of the complex of cytochrome oxidase with azide in the presence and absence of cyanide. CcO+N₃, the spectrum of 800 μm CcO in the presence of 700 μm NaN₃ at pH 7.8. CcO+N₃+CN, the spectrum of the same sample of CcO after reaction with 1 mm NaCN.

FIGURE 5.

FTIR spectra of cytochrome oxidase in the presence of both azide and cyanide and after gel filtration. Top, the spectrum of 800 μm CcO in the presence of both 800 μm NaN₃ and 1 mm NaCN at pH 7.8. CcO.N₃.CN/filtration, the spectrum of the same sample of CcO after filtration. CcO.CN/filtration, the spectrum of the complex of CcO with cyanide after gel filtration.

FIGURE 6.

Dependence of the g = 3 EPR signal of oxidized heme a on the presence of azide and cyanide. Control, the control spectrum of 25 μm oxidized CcO at pH 7.8. N₃+CN, in the presence of both 500 μm NaN₃ and 5 mm NaCN. Filtration, the spectrum after filtration. For the conditions of measurements, see “Experimental Procedures.”

FIGURE 7.

Schematic of the transitions at the catalytic site during the reaction of azide and cyanide with oxidized cytochrome oxidase. In the catalytic center of oxidized CcO (O), represented by the large box, water is assumed to be a bridging ligand between Fe_a3³⁺ and Cu_B²⁺. The maximum of the Soret band at 424 nm reflects the high spin state of Fe_a3³⁺. This catalytic site is only accessible to external ligands through a hydrophobic channel connecting Cu_B with the solvent. This channel is depicted as the small box on the right of Cu_B. Ligands in the neutral protonated form diffuse to the binuclear center (54), but they are bound as anions. Azide ion at low concentration displaces water between Fe_a3 and Cu_B. This substitution, however, does not change the spin state of Fe_a3³⁺, and the alteration of the optical spectrum is negligible. Presumably this azide exhibits two modes of the binding indicated by two bands in the FTIR spectra, at 2040 and 2051 cm⁻¹. (O.N3), the addition of cyanide to the preformed azide-oxidase adduct results in the rapid formation of the ternary complex (O.N3.CN). This formation is associated with the appearance of a new azide stretch at 2033 cm⁻¹ and a red shift of the Soret maximum to 428 nm. The strong binding of cyanide to Cu_B blocks the channel that restricts a release of azide from the catalytic center.