Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments (original) (raw)
Related papers
Journal of Biological Chemistry, 2003
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 -CO complex demonstrate that in the presence of 60 -80 M O 2 , the (C-O) of Cu B 1؉ -C-O at 2053 cm ؊1 (complex A) shifts to 2045 cm ؊1 and remains unchanged in H 2 O/D 2 O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a 3 -CO (complex B), however, remain unchanged. The change in the (C-O) of complex A results in an increase of k ؊2 , and thus in a higher affinity of Cu B for exogenous ligands.
Conserved Glycine 232 in the Ligand Channel of ba 3 Cytochrome Oxidase from Thermus thermophilus
Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O 2 and NO with the fully reduced G232V mutant of ba 3 cytochrome c oxidase from Thermus thermophilus (Tt ba 3 ) in which a conserved glycine residue in the O 2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa 3 cytochrome c oxidase suggested that the valine completely blocked the access of O 2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O 2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O 2 and NO binding are not significantly affected in the Tt ba 3 G232V mutant. Classical molecular dynamics simulations of diffusion of O 2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O 2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.
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.
Docking Site Dynamics of ba3-Cytochrome c Oxidase from Thermus thermophilus
Journal of Biological Chemistry, 2003
Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba 3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved stepscan Fourier transform infrared difference spectra of photodissociated CO from heme a 3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to Cu B but is trapped within a docking site near the ring A of heme a 3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (CO ؍ 2131 cm ؊1 , t d ؍ 10-35 s and CO ؍ 2146 cm ؊1 , t d ؍ 85 s) are observed. At later times (t d ؍ 110 s) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.
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.
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.
Journal of Bioenergetics and Biomembranes, 1993
Cytochromeaa 3 ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea 3 (oro)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.
Structural basis for functional properties of cytochromecoxidase
bioRxiv (Cold Spring Harbor Laboratory), 2023
Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis 1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes 2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation 2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) 4 , we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state 5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO. Main Mammalian CcO is a large integral membrane protein comprised of 13 subunits. It contains four redox active centers, CuA, heme a, and a heme a3/CuB binuclear center (BNC) (Fig. 1A). Molecular oxygen binds to the heme a3 iron in the BNC, where it is reduced to two water molecules by accepting four electrons from cytochrome c and four protons (the "substrate" protons) from the negative side (N-side) of the mitochondrial membrane (Fig. 1A). The energy derived from the oxygen reduction chemistry is used to drive the translocation of four protons (the "pumped" protons) from the N-side to the positive side (P-side) of the membrane 1,7. Strong evidence suggests that the substrate protons are delivered to the BNC via the D and K-channel (see Extended Data