Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of carbon monoxide photodissociation intermediates

Abstract

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.

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus

“…2, traces A-B and B-G). The ratio of the relative areas of the Fe-CO/Cu-CO is 4:1 in the absolute spectra, and considering the integrated absorptivities expected of CO complexes of heme and copper proteins (26), the ratio of ⑀ Fe-CO /⑀ Cu-CO is ϳ1.5:1 suggesting ϳ30% of unligated heme a 3 , in agreement with the magnetic circular dichroism (17) and optical data (19). 13 CO; E, pD 6.5; F, pD 8.5; and G, pD 9.7.…”

“…This indicates that the flow-flash experimental approach, in which the loss of CO is the rate-determining step in the formation of the heme-O 2 adduct and thus requires the photodissociated CO not to interfere with the reaction with O 2 , is not the appropriate method for the identification of oxygen intermediates during the catalytic turnover of the ba 3 enzyme (39). It has been reported that Cu B in ba 3 , in contrast to bovine aa 3 , has a relative high affinity for CO (K 1 Ͼ 10 4 ) indicating unusual kinetics of electron transfer and ligand binding (17,19). The kinetic properties of CO and CN binding were interpreted, prior to the determination of the threedimensional structure of the enzyme to peculiar features in the binuclear center (19).…”

“…where k 1 and k Ϫ1 represent the reversible binding of CO to Cu B , and k 2 is the first-order transfer of CO from Cu B to the heme-Fe (12)(13)(14)(15)(16)(17)(18)(19)(20). The thermal dissociation rate of the heme-CO complex of bovine fully reduced cytochrome c oxidase-CO is very slow (0.023 s Ϫ1 ) and thus, k Ϫ2 can be neglected (13).…”

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

“…In particular, binding of CO to this enzyme leads to formation of only 70% of the heme a 3 -CO complex (21,24), with the remaining 30% forming a CuB-CO complex (25). To estimate the extent of NO binding to heme a 3 , the inset of Figure 1B shows the second derivative of the Soret spectra of the unligated and NO-bound forms.…”

Spectroscopic and Kinetic Investigation of the Fully Reduced and Mixed Valence States of ba3-Cytochrome c Oxidase from Thermus thermophilus

“…In the case of fully reduced cytochrome ba 3 , the heme a 3 -Cu B binuclear center is subjected to peculiar properties (53)(54)(55)(56)(57) characterized by a high affinity of Cu B for CO (K Ͼ 10 4 M Ϫ1 ) and a slow intramolecular ligand transfer to heme a 3 (k ϭ 8 s Ϫ1 ). As a result, cytochrome ba 3 is the only documented oxidase where the binding of CO to Cu B is exergonic and accounts for 25-30% of the total enzyme concentration (34).…”