Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (1l is) as the postphotodissociation Cu'-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CUB to Fe,3 when CO binds to CUB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The timeresolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CUB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fe,3 pair. We speculate as to the identity ofthe ligand that is transferred between CUB and Fe,13 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.In a recent time-resolved infrared (TRIR) study (1) of the events after photodissociation of CO from cytochrome (cyt) a3 of reduced beef heart cytochrome oxidase (CcO), we reported conclusive evidence that photodissociated CO binds quantitatively to CuB at room temperature prior to equilibrating with solution. In a parallel kinetics study (6.E., P. M.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.

Nanosecond time-resolved magnetic circular dichroism (TRMCD) and time-resolved natural circular dichroism (TRCD) measurements of photolysis products of the CO complex of eukaryotic cytochrome c oxidase (CcO-CO) are presented. TRMCD spectra obtained at 100 ns and 10 microseconds after photolysis are diagnostic of pentacoordinate cytochrome a3Fe2+, as would be expected for simple photodissociation. Other time-resolved spectroscopies (UV-visible and resonance Raman), however, show evidence for unusual Fea3(2+) coordination after CO photolysis (Woodruff, W. H., O. Einarsdóttir, R. B. Dyer, K. A. Bagley, G. Palmer, S. J. Atherton, R. A. Goldbeck, T. D. Dawes, and D. S. Kliger. 1991. Proc. Nat. Acad. Sci. U.S.A. 88:2588-2592). Furthermore, time-resolved IR experiments have shown that photodissociated CO binds to CuB+ prior to recombining with Fea3(2+) (Dyer, R. B., O. Einarsdóttir, P. M. Killough, J. J. López-Garriga, and W. H. Woodruff. 1989. J. Am. Chem. Soc. 111:7657-7659). A model of the CcO-CO photolysis cycle which is consistent with all of the spectroscopic results is presented. A novel feature of this model is the coordination of a ligand endogenous to the protein to the Fe axial site vacated by the photolyzed CO and the simultaneous breaking of the Fe-imidazole(histidine) bond.

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Electron transfer following photolysis of CO from miXed-valence (Cytochrome a3+ Cu2+ cytochrome a2+-CO CuB) cytochrome oxidase (ferrocytochrome-c; oxygen oxidoreductase, EC 1.9.3.1) was studied on time scales of nanoseconds to milliseconds by mutchannel time-resolved optical absorption spectroscopy. In this method, the optical absorption was measured at many wavelengths simultaneously by using an optical spectrometric multichannel analyzer system. The highquality time-resolved difference spectra showed a large increase on a microsecond time scale in the visible region centered at -520 nm and in the UV region centered at =390 am. These absorbance changes were not observed after photodissociation of CO from the fully reduced enzyme and therefore are attributed to intramolecular electron transfer. Simultaneously, there was a blue shift and a small increase in the a band, which is attributed to the reduction of cytochrome a. Approximately one-third of the absorbance change at 520 am can be attributed to reduction of cytochrome a. The absorbance changes associated with the 520-and the 390-am bands are on the same time scale (t1/2 2 us) as the dissociation of CO from CuBreported previously by time-resolved infrared spectroscopy. The position and shape of these bands are reasonable for chargetranser transitions involving copper(U). We suggest that the absorbance increase at 520 am, which cannot be attributed to a reduction of cytochrome a, may represent a charge transfer involving CUI+ accompanying the oxidation of CU+ to CUB+.The absorbance increase at 390 am is also partially attributed to this transition. These results suggest that Cu2B+ may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.Cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1), the terminal oxidase of cellular respiration, catalyzes the four-electron transfer from cytochrome c to molecular oxygen. The enzyme contains two heme A chromophores (cytochromes a and a3) and two redox-active coppers (CUA and CUB). The energy that is derived from the reduction of dioxygen to water is coupled to active translocation of protons across the membrane and used by the cell to synthesize ATP, the ubiquitous biological energy source (1). Of the four redox active centers, cytochrome a and CUA are believed to be the primary acceptors of electrons from cytochrome c, followed by electron transfer to the site of 02 reduction, which comprises cytochrome a3 and CUB. However, the details of the mechanism ofthe dioxygen reduction, how the enzyme functions as a proton pump, and how the proton pumping is linked to electron transfer remain unclear. Adequate understanding of the precise electron-transfer sequence among the four redox-active metal centers would clearly help to elucidate the role of the individual redox centers as electron carriers and perhaps to increase our understanding of how electron transfer may be coupled to proton translocation. Electron transfer in cytochrome oxidase can be studied by phot...