Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus (original) (raw)
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Biochemistry, 1999
The ba 3 cytochrome c oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS, and FTIR spectroscopic approach. Oxidative electrochemical redox titrations yielded midpoint potentials of E m 1 ) -0.02 ( 0.01 V and E m 2 ) 0.16 ( 0.04 V for heme b and E m 1 ) 0.13 ( 0.04 V and E m 2 ) 0.22 ( 0.03 V for heme a 3 (vs Ag/AgCl/3 M KCl). Fully reversible electrochemically induced UV/VIS and FTIR difference spectra were obtained for the full potential step from -0.5 to 0.5 V as well as for the critical potential steps from -0.5 to 0.1 V (heme b is fully oxidized and heme a 3 remains essentially reduced) and from 0.1 to 0.5 V (heme b remains oxidized and heme a 3 becomes oxidized). The difference spectra thus allow to us distinguish modes coupled to heme b and heme a 3 . Analogous difference spectra were obtained for the enzyme in D 2 O buffer for additional assignments. The FTIR difference spectra reveal the reorganization of the polypeptide backbone, perturbations of single amino acids and of hemes b and a 3 upon electron transfer to/from the four redoxactive centers heme b and a 3 , as well as Cu B and Cu A . Proton transfer coupled to redox transitions can be expected to manifest in the spectra. Tentative assignments of heme vibrational modes, of individual amino acids, and of secondary structure elements are presented. Aspects of the uncommon electrochemical and spectroscopic properties of the ba 3 oxidase from T. thermophilus are discussed.
Journal of Biological Chemistry, 2011
Elucidating the properties of the heme Fe-Cu B binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the 7a (CO) of the protonated form of Tyr residues at 1247 cm ؊1 and that of the deprotonated form at 1301 cm ؊1 . By monitoring the intensity changes of the 1247 and 1301 cm ؊1 modes as a function of pH, we measured a pK a of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860 -32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ϳ607 nm P (Fe(IV)؍O) species in the pH 6 -9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a 3 ring D propionate-H 2 O-ring A propionate-Asp-372 site to the exit/ output proton channel (H 2 O pool) is discussed.
Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
The respiratory heme-copper oxidases catalyze reduction of O 2 to H 2 O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa 3-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba 3 oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H + / electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A-and the B-class ba 3 oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
The oxidative part of the catalytic cycle of the caa 3 -type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa 3 -type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa 3 oxidases in general and to ba 3 oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu A and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O H state into the final product of the reaction-E H (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa 3 -type enzymes, we concluded that the finally produced E H state of caa 3 oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O H → E H transition of the aa 3 -type oxidases. So, the fully-reduced caa 3 oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.
Kinetic Properties of ba 3 Oxidase from Thermus thermophilus : Effect of Temperature †
Biochemistry, 1999
The kinetic properties of the ba 3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70°C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O 2 reaction, the decay of the F intermediate is significantly slower (k′) 100 s-1 at 5°C) than in the mitochondrial enzyme, with an activation energy E* of 10.1 (0.9 kcal mol-1. The cyanide-inhibited ba 3 oxidizes cyt c 522 quickly (k ≈ 5 × 10 6 M-1 s-1 at 25°C) and selectively, with an activation energy E* of 10.9 (0.9 kcal mol-1 , but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c 552 oxidase activity is enhanced up to 60°C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c 522-ba 3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba 3 for CO at 20°C (K eq) 1 × 10 5 M-1) was found to be smaller than that of beef heart aa 3 (K eq) 4 × 10 6 M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a 3 2+ of ba 3 (k′) 0.8 s-1 vs k′) 0.02 s-1 for beef heart aa 3 at 20°C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O 2 tensions is discussed.
Biochemistry, 2010
Cytochrome ba 3 from T. thermophilus is a member of the B-type haem-copper oxidases, which have low sequence homology to the well-studied mitochondrial-like A-type. Recently, it was suggested that the ba 3 oxidase has only one pathway for proton delivery to the active site, and that this pathway is spatially analogous to the K-pathway in the A-type oxidases. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the timeresolved reaction between fully reduced cytochrome ba 3 and O 2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312→Ser variant in the ba 3 oxidase both reactions associated with proton uptake from solution, the P R →F and F→O transitions, were slowed compared to the wild-type ba 3 . The observed time constants were slowed ~3-fold (P R →F, to ~170 μs from 60 μs in wild-type) and ~30-fold (F→O, to ~40 ms from 1.1 ms). In the Thr-315→Val variant, the F→O transition was about 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P R →F transition displayed an essentially unchanged time constant. However, proton uptake from solution was a factor of two slower and decoupled from the optical P R →F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, in strong support for the suggested involvement of the T312 and T315 in proton transfer to the active site during O 2 reduction in the ba 3 oxidase.
The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
Cytochrome ba 3 (ba 3) of Thermus thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a 3) and a copper (Cu B). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba 3 with O 2 using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s − 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O 2 binds rapidly to heme a 3 in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O 2 binds transiently to a trap, likely Cu B , prior to its migration to heme a 3 for the oxidative reaction, highlighting the critical role of Cu B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.
Accounts of Chemical Research, 2019
CONSPECTUS: Cytochrome c oxidase (CcO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven CcO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba 3 oxidase is a Btype oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O 2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other CcOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS 2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba 3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O 2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of Cu B for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba 3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba 3 oxidase into a high O 2affinity enzyme that functions effectively in its extreme native environment. The present results show that ba 3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba 3 oxidase.
Single Mutations That Redirect Internal Proton Transfer in the ba3 Oxidase from Thermus thermophilus
Biochemistry, 2013
The ba 3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa 3-type oxidases proton uptake to the catalytic site and "pump site" occur simultaneously. However, with the ba 3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than with the aa 3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba 3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though the ba 3 CytcO uses only a single proton pathway for transfer of the substrate and "pumped" protons, the aminoacid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site, respectively. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.