Pulse Radiolysis Studies of Temperature Dependent Electron Transfers among Redox Centers in ba3-Cytochrome c Oxidase from Thermus thermophilus: Comparison of A- and B-Type Enzymes.

The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the CuAr site to the low-spin heme-(a)bo site, i.e., CuAr + heme-a(b)o → CuAo + heme-a(b)r in three structurally characterized enzymes: A-type aa3 from Paracoccus denitrificans (PDB code 3HB3) and bovine heart tissue (PDB code 2ZXW), and the B-type ba3 from T. thermophilus (PDB codes 1EHK and 1XME). k,T data sets were obtained with the use of pulse radiolysis as described previously. Semiclassical Marcus theory revealed that λ varies from 0.74 to 1.1 eV, Hab, varies from ∼2 × 10-5 eV (0.16 cm-1) to ∼24 × 10-5 eV (1.9 cm-1), and ßD varies from 9.3 to 13.9. These parameters are consistent with diabatic electron tunneling. The II-Asp111Asn CuA mutation in cytochrome ba3 had no effect on the rate of this reaction whereas the II-Met160Leu CuA-mutation was slower by an amount corresponding to a decreased driving force of ∼0.06 eV. The structures support the presence of a common, electron-conducting "wire" between CuA and heme-a(b). The transfer of an electron from the low-spin heme to the high-spin heme, i.e., heme-a(b)r + heme-a3o → heme-a(b)o + heme-a3r, was not observed with the A-type enzymes in our experiments but was observed with the Thermus ba3; its Marcus parameters are λ = 1.5 eV, Hab = 26.6 × 10-5 eV (2.14 cm-1), and ßD = 9.35, consistent also with diabatic electron tunneling between the two hemes. The II-Glu15Ala mutation of the K-channel structure, ∼ 24 Å between its CA and Fe-a3, was found to completely block heme-br to heme-a3o electron transfer. A structural mechanism is suggested to explain these observations.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase.

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

Interactions of Cu(B) with Carbon Monoxide in Cytochrome c Oxidase: Origin of the Anomalous Correlation between the Fe-CO and C-O Stretching Frequencies.

In heme-copper oxidases, the correlation curve between the iron-CO and C-O stretching vibrational modes (ν(Fe-CO) and ν(C-O), respectively) is anomalous as compared to the correlation in other heme proteins. To extend the correlation curve, the resonance Raman (RR) and infrared (IR) spectra of the CO adducts of cytochrome ba3 (ba3) from Thermus thermophilus were measured. The RR spectrum has two strong ν(Fe-CO) lines (508 and 515 cm(-1)) and a very weak line at 526 cm(-1), and the IR spectrum has three ν(C-O) lines (1966, 1973, and 1981 cm(-1)), indicating the presence of multiple conformers. Employing photodissociation methods, the ν(Fe-CO) RR and ν(C-O) IR lines were assigned to each conformer, enabling the establishment of a reliable inverse correlation curve for the ν(Fe-CO) versus the ν(C-O) stretching frequencies. To determine the molecular basis of the correlation, a series of DFT calculations on 6-coordinate porphyrin-CO compounds and a model of the binuclear center of the heme-copper oxidases were carried out. The calculations demonstrated that the copper unit model caused significant mixing among porphyrin-CO molecular orbitals (MOs) that contribute to the Fe-C and C-O bonding interactions, and also indicated the presence of mixing between the d(z)(2) orbital of the copper and MOs that are responsible for the ν(Fe-CO) vs ν(C-O) inverse correlation. Together, the spectroscopic and DFT results clarify the origin of the anomaly of ν(Fe-CO) and ν(C-O) frequencies in the heme-copper oxidases, a long-standing issue.

Linking chemical electron-proton transfer to proton pumping in cytochrome c oxidase: broken-symmetry DFT exploration of intermediates along the catalytic reaction pathway of the iron-copper dinuclear complex.

After a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome c oxidase, we present the results of our earlier and recent density functional theory calculations for the dinuclear Fe-a3-CuB reaction center in this enzyme. A specific catalytic reaction wheel diagram is constructed from the calculations, based on the structures and relative energies of the intermediate states of the reaction cycle. A larger family of tautomers/protonation states is generated compared to our earlier work, and a new lowest-energy pathway is proposed. The entire reaction cycle is calculated for the new smaller model (about 185-190 atoms), and two selected arcs of the wheel are chosen for calculations using a larger model (about 205 atoms). We compare the structural and redox energetics and protonation calculations with available experimental data. The reaction cycle map that we have built is positioned for further improvement and testing against experiment.

Conserved glycine 232 in the ligand channel of ba3 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 O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617-11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 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 O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases.

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

Mobility of Xe atoms within the oxygen diffusion channel of cytochrome ba(3) oxidase.

We use a form of "freeze-trap, kinetic crystallography" to explore the migration of Xe atoms away from the dinuclear heme a(3)/Cu(B) center in Thermus thermophilus cytochrome ba(3) oxidase. This enzyme is a member of the heme-copper oxidase superfamily and is thus crucial for dioxygen-dependent life. The mechanisms involved in the migration of oxygen, water, electrons, and protons into and/or out of the specialized channels of the heme-copper oxidases are generally not well understood. Pressurization of crystals with Xe gas previously revealed a O(2) diffusion channel in cytochrome ba(3) oxidase that is continuous, Y-shaped, 18-20 Šin length and comprised of hydrophobic residues, connecting the protein surface within the bilayer to the a(3)-Cu(B) center in the active site. To understand movement of gas molecules within the O(2) channel, we performed crystallographic analysis of 19 Xe laden crystals freeze-trapped in liquid nitrogen at selected times between 0 and 480 s while undergoing outgassing at room temperature. Variation in Xe crystallographic occupancy at five discrete sites as a function of time leads to a kinetic model revealing relative degrees of mobility of Xe atoms within the channel. Xe egress occurs primarily through the channel formed by the Xe1 → Xe5 → Xe3 → Xe4 sites, suggesting that ingress of O(2) is likely to occur by the reverse of this process. The channel itself appears not to undergo significant structural changes during Xe migration, thereby indicating a passive role in this important physiological function.

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus.

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from Thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a(3), and for Glu126(II) (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126(II), and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a(3), was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a(3) or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a(3) is a good candidate to be the proton loading site.

Electrochemical and infrared spectroscopic analysis of the interaction of the Cu(A) domain and cytochrome c(552) from Thermus thermophilus.

The hydrophobically guided complex formation between the Cu(A) fragment from Thermus thermophilus ba(3) terminal oxidase and its electron transfer substrate, cytochrome c(552), was investigated electrochemically. In the presence of the purified Cu(A) fragment, a clear downshift of the c(552) redox potential from 171 to 111mV±10mV vs SHE' was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B).

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2Å resolution, reveal a Fe-C distance of ~2.0Å, a Cu-O distance of 2.4Å and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9Å and an O-Fe distance of ~2.3Å. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2Å and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.

The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus.

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 (~1ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k=400s(-1)) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1ms 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.

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment.

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H(+) and e(-) transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba(3)-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O(2)-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu(B) atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe(a3) and Cu(B) atoms that is best modeled as peroxide. The structure of ba(3)-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba(3)-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus.

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba(3) cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O(2)-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O(2)-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba(3) simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O(2) binding to heme a(3) at 90 µM O(2) occurs with lifetimes of 9.3 and 110 µs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 µs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 10(9) M(-1) s(-1) for O(2) binding to ba(3) in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O(2) bond cleavage in ba(3) of 4.8 µs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O(2) to the active site in ba(3) and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a(3) site in ba(3), making the CO flow-flash method inapplicable.

Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba3 Cytochrome c oxidase from Thermus thermophilus.

Cytochrome ba(3) from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169-16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved 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 those of wild-type ba(3). The observed time constants were slowed approximately 3-fold (for P(R) --> F, from 60 to approximately 170 mus in the wild type) and approximately 30-fold (for F --> O, from 1.1 to approximately 40 ms). In the Thr-315 --> Val variant, the F --> O transition was approximately 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P(R) --> F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 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, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O(2) reduction in the ba(3) oxidase.

The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping.

The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B- and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D- and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba(3) oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the Cu(B) and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.

Accommodation of two diatomic molecules in cytochrome bo: insights into NO reductase activity in terminal oxidases.

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N(2)O. Previously, we characterized the heme a(3)-NO complex formed in cytochrome ba(3) from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu(B)(I) to form an end-on NO-Cu(B) or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo(3) from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N(2)O. The EPR and resonance Raman signatures of the heme o(3)-NO complex are comparable to those of the a(3)-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o(3)-NO complex does not produce a Cu(B)-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o(3)-NO.OC-Cu(B)] tertiary complex can form in bo(3) but not in ba(3). We assign these differences to a greater iron-copper distance in the reduced form of bo(3) compared to that of ba(3). Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu(B) is not an essential step of the reaction mechanism.

Combined microspectrophotometric and crystallographic examination of chemically reduced and X-ray radiation-reduced forms of cytochrome ba3 oxidase from Thermus thermophilus: structure of the reduced form of the enzyme.

Three paths for obtaining crystals of reduced (II-E4Q/I-K258R) cytochrome ba(3) are described, and the structures of these are reported at approximately 2.8-3.0 A resolution. Microspectrophotometry of single crystals of Thermus ba(3) oxidase at 100 K was used to show that crystals of the oxidized enzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron Radiation Laboratory), being nearly complete in 1 min. The previously reported structures of ba(3) (Protein Data Bank entries 1EHK and 1XME ), having a crystallographically detectable water between the Cu(B) and Fe(a3) metals of the dinuclear center, actually represent the X-ray radiation-reduced enzyme. Dithionite-reduced crystals or crystals formed from dithionite-reduced enzyme revealed the absence of the above-mentioned water and an increase in the Cu(B)-Fe(a3) distance of approximately 0.3 A. The new structures are discussed in terms of enzyme function. An unexpected optical absorption envelope at approximately 590 nm is also reported. This spectral feature is tentatively thought to arise from a five-coordinate, low-spin, ferrous heme a(3) that is trapped in the frozen crystals.

Toward a chemical mechanism of proton pumping by the B-type cytochrome c oxidases: application of density functional theory to cytochrome ba3 of Thermus thermophilus.

A mechanism for proton pumping by the B-type cytochrome c oxidases is presented in which one proton is pumped in conjunction with the weakly exergonic, two-electron reduction of Fe-bound O 2 to the Fe-Cu bridging peroxodianion and three protons are pumped in conjunction with the highly exergonic, two-electron reduction of Fe(III)- (-)O-O (-)-Cu(II) to form water and the active oxidized enzyme, Fe(III)- (-)OH,Cu(II). The scheme is based on the active-site structure of cytochrome ba 3 from Thermus thermophilus, which is considered to be both necessary and sufficient for coupled O 2 reduction and proton pumping when appropriate gates are in place (not included in the model). Fourteen detailed structures obtained from density functional theory (DFT) geometry optimization are presented that are reasonably thought to occur during the four-electron reduction of O 2. Each proton-pumping step takes place when a proton resides on the imidazole ring of I-His376 and the large active-site cluster has a net charge of +1 due to an uncompensated, positive charge formally associated with Cu B. Four types of DFT were applied to determine the energy of each intermediate, and standard thermochemical approaches were used to obtain the reaction free energies for each step in the catalytic cycle. This application of DFT generally conforms with previously suggested criteria for a valid model (Siegbahn, P. E. M.; Blomberg, M. A. R. Chem. Rev. 2000, 100, 421-437) and shows how the chemistry of O 2 reduction in the heme a 3 -Cu B dinuclear center can be harnessed to generate an electrochemical proton gradient across the lipid bilayer.

Electron and proton transfer in the ba(3) oxidase from Thermus thermophilus.

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.