Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Abstract

Elucidating the structures of intermediates in the reduction of O2 to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O2 with the mixed-valence enzyme, in which only heme a3 and CuB in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O==O bond cleavage occurs within the first 200 micros after reaction initiation; the presence of a uniquely stable Fe---O---O(H) peroxy species is not detected. The product of this rapid reaction is a heme a3 oxoferryl (FeIV==O) species, which requires that an electron donor in addition to heme a3 and CuB must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A. , Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547-10553] show that one of the CuB ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O---O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His---Tyr species to produce the product oxoferryl species, CuB2+---OH-, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O---O bond forming chemistry that occurs during O2 evolution in photosynthesis.

Figure 1

Experimental apparatus. ENZ, enzyme solution; BUF, O₂-saturated buffer; MIX, active mixer; JCK, thermostated jacket; FC, flow cell; LB, laser beams; L1, L2, L3, lenses; F1, notch filter; F2, short-pass filter; F3, depolarizer; SL, entrance slit; POL, polychromator; CCD, CCD detector.

Figure 2

High-frequency Raman spectra of mixed-valence (a) and fully reduced (b) CcO at 10 ns after photolysis of CO. Trace a is an average of several 10-ns spectra obtained as described in Materials and Methods. Trace b was obtained in a separate experiment under the same conditions as used for trace a; the CO complex of the fully reduced CcO was prepared by reduction with 2 mM ascorbate/1 μM cytochrome c under a CO atmosphere.

Figure 3

Reaction between MV-CcO and O₂ at 20^oC. Difference Raman spectra were calculated at the indicated times after initiation of the reaction between MV-CcO and O₂ by subtracting the spectrum recorded with ¹⁸O₂ as a substrate from that obtained with ¹⁶O₂. All porphyrin vibrations cancel in the difference spectra, whereas vibrations of bound oxygen appear as derivative-shaped features. Full scale of the ordinate axis is 28% of the porphyrin ν₇ mode in the absolute spectra. Each trace is an average of several isotope-difference spectra (1–1.5 × 10⁶ shots per isotope total) obtained in independent experiments.

Figure 4

Time course of the reaction between MV-CcO and O₂ at 20^oC. Relative intensities of the 568/545 cm⁻¹ (○) and 804/768 cm⁻¹ (•) difference bands were extracted from the data set in Fig. 3. Solid lines represent the results of the kinetic fitting procedure described in the text. Error bars denote uncertainty in the intensities from the signal/noise ratio in the corresponding difference spectra.

Figure 5

A model for dioxygen bond cleavage by MV-CcO. Only the chemically essential sites (heme a₃, Cu_B, His240, and Tyr244) are shown for clarity. Brackets denote transition states; details are given in the text.