Oxygen-linked Equilibrium CuB-CO Species in Cytochrome ba3 Oxidase from Thermus thermophilus

Abstract

We report the first study of O 2 migration in the putative O 2 channel of cytochrome ba 3 and its effect to the properties of the binuclear heme a 3 -Cu B center of cytochrome ba 3 from Thermus thermophilus. The Fourier transform infrared spectra of the ba 3 Cytochrome ba 3 from Thermus thermophilus is a member of the large family of structurally related heme-copper oxidases (1, 2). It catalyzes both the four-electron reduction of O 2 to H 2 O, converting the energy of this reaction to a transmembrane proton motive force, and the two-electron reduction of NO to N 2 O (1-4). Based on the crystal structure, the enzyme contains a homodinuclear copper (Cu A ), a low-spin heme b, and a heme a 3 -Cu B binuclear center (1). The ba 3 -oxidase retains the electron transport chain functional under low oxygen concentration in the medium. In both structurally characterized aa 3 -type heme-copper oxidases, three possible O 2 channels have been suggested (5, 6). One of them leads from a trapped lipid pocket in subunit III to the active site and contains Glu-278 (Paracoccus denitrificans numbering), a residue near Cu B (5, 6). In the presence of three O 2 channels in aa 3 -type oxidases, it is not known yet whether a facilitated oxygen channel is necessary, because the O 2 concentrations normally far exceed the O 2 affinity of the enzyme; thus, excess O 2 would not be rate-limiting. The proposed oxygen input channel in ba 3 contains Ile-235 instead of Glu-278 (conserved to aa 3 oxidases), which optimizes the formation of a hydrophobic pore (1, 2). The evolutionary development of an optimized oxygen channel is appropriate for organisms such as T. thermophilus ba 3 oxidase, the archaeal Sulfolobus acidocaldarius aa 3 -quinol oxidase, and Natronomonas pharaonics, which grow under low oxygen tension and at high temperatures (2). However, no data exist in the literature to demonstrate the nature of the conformational changes that occur in the binuclear center in the presence of O 2 in the channel. Because of the unusual ligand-binding and kinetic properties of the binuclear center, cytochrome ba 3 oxidase is unique among the heme-copper oxidases in that it is susceptible to a detailed kinetic analysis of its ligand dynamics (4, 7). The binding of CO to the binuclear center of ba 3 follows that found in all heme-copper oxidases and proceeds according to the Scheme 1 (7-12).In our previous work (7), we identified the C-O stretching mode of the equilibrium Cu B 1ϩ -CO species (complex A) at 2053 cm Ϫ1 and concluded that the environment in the binuclear center does not alter the protonation state of the Cu B histidine ligands. Understanding the conformational transitions that are associated with protonation/deprotonation of labile residues is essential because ionizable groups whose pK a values are near physiological pH are involved in proton uptake or release. A hydrogen-bonded connectivity between the propionates of heme a 3 , Asp-372, and H 2 O was also reported. Accordingly, plausible mechanisms of proton pathway(s) directly assoc...

Simultaneous Resonance Raman Detection of the Heme a3-Fe-CO and CuB-CO Species in CO-bound ba3-Cytochrome c Oxidase from Thermus thermophilus

“…Given that the binuclear centers in ba 3 and its counterpart caa 3 oxidase are very similar, the same argument holds for the absence of the ␤-form in ba 3 oxidase but its presence in its counterpart caa 3 . Taken together, the present data and data previously reported (4,5,9,21) demonstrate that fully active heme-copper oxidases can have binuclear centers in which either the ␣-or the ␤-form is present.…”

“…The insensitivity of the (CO) of Cu B frequency to H 2 O/D 2 O exchange and to pH/pD 5.5-9.7 range indicated that the degree of back-donation of electron density from the d orbitals to the antibonding * orbitals is not altered under these conditions (5,8). In the presence of small amounts of O 2 , however, the (C-O) of Cu B 1ϩ -CO at 2053 cm Ϫ1 (complex A) shifts to 2045 cm Ϫ1 and remains unchanged in H 2 O/D 2 O exchanges and in the pH 6.5-9.0 range (5).…”

“…Possible candidates included the cross-linked, conserved tyrosine that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu B . We have recently demonstrated that that the absence of the farnesyl-tyrosine linkage has no direct control on the (CO) stretching frequency and thus on the type (␣ or ␤) of the stable conformation that is present in heme-copper oxidases (4,5,9,21). It has also been established that the Cu B -His environment is very rigid and not subject to conformational transitions that are associated with protonation/deprotonation events of the Cu B His ligands (5).…”

“…We have recently demonstrated that that the absence of the farnesyl-tyrosine linkage has no direct control on the (CO) stretching frequency and thus on the type (␣ or ␤) of the stable conformation that is present in heme-copper oxidases (4,5,9,21). It has also been established that the Cu B -His environment is very rigid and not subject to conformational transitions that are associated with protonation/deprotonation events of the Cu B His ligands (5). The binuclear centers of ba 3 (␣-form) and caa 3 (␤-form) oxidases contain the conserved tyrosine; therefore, we can exclude that the crosslinked tyrosine provides the ionizable group that causes the heme-Cu B distance to change.…”

“…Cytochrome ba 3 contains a homodinuclear copper complex (Cu A ), one low spin heme b, and a binuclear center that consists of Cu B and heme a 3 (1). The comparative kinetics data on CO photodissociation and rebinding of various heme-copper oxidases and the derived activation parameters have indicated that the COligation/release mechanism in cytochrome ba 3 follows that found in other heme-copper oxidases (3)(4)(5)(6)(7)(8)(9) and proceeds according to the following scheme. In contrast to the bovine aa 3 oxidase, Cu B of cytochrome ba 3 has a relative high affinity for CO (k 1 Ͼ 10 4 ), whereas the transfer of CO to heme a 3 2ϩ is characterized by a small k 2 ϭ 8 s Ϫ1 and by a k Ϫ2 ϭ 0.8 s Ϫ1 that is 30-fold greater than that of the bovine aa 3 (3,4).…”