Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans

Abstract

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.

Fig. 1.

Schematic catalytic cycle and artificial intermediates of CcO. (A) The fully oxidized O state is successively reduced to the E and then to the R state. The R state binds molecular oxygen resulting in the transient A state. Oxygen receives four electrons simultaneously from the protein resulting in complete reduction (P state). Receiving the third electron leads to building of the F state. The fourth electron completes the cycle by regenerating the O state. (B) The P state can be formed artificially by treating O state CcO with either one to five equivalents of hydrogen peroxide at pH 9 or with carbon monoxide in the presence of molecular oxygen at pH > 7. The F state can be reached by treatment with an excess (we used 500 equivalents) of H₂O₂. This F state can be transformed, as shown in this study, by treatment with ammonia at pH 9 to the previously undescribed P_N state.

Fig. 2.

Difference absorption spectra (induced states minus O state) of artificial CcO intermediates at pH 9. The P_H (blue curve) and F state (red curve) were prepared by addition of 5 and 500 equivalents hydrogen peroxide, respectively, and the P_CO state (violet curve) was induced by aeration of the O state with CO gas. Treatment of the F state with ammonia resulted in formation of the P_N state (green curve). The characteristic difference maxima of the α-band (600 nm in the O state) indicate the formation of an oxoferryl state. Soret bands (O state: 426 nm) are red-shifted compared to the O state noticeably by the minima and maxima in the Soret region. Absorption differences in the Soret region of the F and P_N states are very similar.

Fig. 3.

α-Band difference spectra (induced states minus O state) of artificial CcO intermediates series. (A and B) 6–8 μM of fully oxidized solubilized CcO in 50 mM glycine pH 9.0 or 50 mM MES pH 6.0 and 0.05% lauryl maltoside (LM) were successively mixed with 5 equivalents of H₂O₂ (blue curve) to induce the P_H state (pH 9.0) or the F^• state (pH 6.0), respectively, followed by addition of 500 equivalents of H₂O₂ (red curve) to form the F state, which was transformed with 20 mM ammonia to the new P_N state (green curve) at pH 9.0. At pH 6.0, no reaction with ammonia was observed until the pH was raised to pH 9 (B, cyan curve). Lowering the pH from 9 to 6 resulted in reverse formation of the F/F^• state (A, cyan curve). (C and D) Time-dependent development of 6–8 μM CcO in the P_N (1∶500 H₂O₂ and 20 mM ammonia) and the F (1∶500 H₂O₂) state after catalase addition.

Fig. 4.

EPR spectra of CcO artificial intermediates. Just as for the absorption difference spectra CcO samples were successively mixed with H₂O₂ and NH₃ at pH 9 and pH 6 to prepare different intermediates. (A) EPR spectra of CcO intermediates recorded at 20 K, 2 mW, and a modulation amplitude of 1 mT at 9.6 GHz microwave frequency. The O state CcO (200 μM in 50 mM glycine pH 9 and 0.05% LM) is shown as the black curve. The P_H (blue solid curve), prepared at pH 9, and F^• (blue dotted curve), prepared at pH 6, show an additional radical signal. The F state (red curve) shows no radical at either pH (pH 9 is shown). Both catalase and ammonia in any order were added to prepare the P_N state (green curve) from the F state, resulting in the reappearance of the Y167 radical at pH 9, which vanishes after lowering the pH to pH 6. Addition of catalase or ammonia individually resulted in no change from the F state. (B) EPR spectra of CcO intermediates were recorded at 20 K, 2 mW, and a modulation amplitude of 0.4 mT at 9.6 GHz microwave frequency. These spectra are difference spectra (spectrum of respective intermediate minus O state spectrum) to study the radical signal in more detail. The P_H state (blue solid curve), prepared at pH 9, and F^• state (blue dotted curve), prepared at pH 6, contain a radical signal that is formed again in the P_N state prepared from the F state at pH 9 with ammonia and catalase (green solid curve) but not at pH 6 (green dotted curve). When the P_N state is prepared at pH 6 and the pH is then raised to pH 9, the same radical signal does reappear (cyan curve).