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Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain that catalyzes respiratory reduction of dioxygen ([O.sub.2]) to water in all eukaryotes and many aerobic bacteria. CcO, and its homologs among the heme-copper oxidases, has an active site composed of an oxygen-binding heme and a copper center in the vicinity, plus another heme group that donates electrons to this site. In most oxidoreduction enzymes, electron transfer (eT) takes place by quantum-mechanical electron tunneling. Here we show by independent molecular dynamics and quantum-chemical methods that the heme-heme eT in CcO differs from the majority of cases in having an exceptionally low reorganization energy. We show that the rate of interheme eT in CcO may nevertheless be predicted by the Moser-Dutton equation if reinterpreted as the average of the eT rates between all individual atoms of the donor and acceptor weighed by the respective packing densities between them. We argue that this modification may be necessary at short donor/ acceptor distances comparable to the donor/acceptor radii. biological electron transfer | heme-copper oxidases | Moser-Dutton ruler | reorganization energy doi/ 10.1073/pnas.1005889107

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.sub.3] cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile [O.sub.2]-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the [O.sub.2]-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which [O.sub.2] binding to heme [a.sub.3] at 90 [micro]M [O.sub.2] occurs with lifetimes of 9.3 and 110 [micro]s in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 [micro]s), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 x [10.sup.9][M.sup.-1][s.sup.-1] for [O.sub.2] binding to ba3 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.sub.2] bond cleavage in [ba.sub.3] of 4.8 [micro]s is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of [O.sub.2] to the active site in ba3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a3 site in [ba.sub.3], making the CO flow-flash method inapplicable. double-laser technique | T. thermophilus [ba.sub.3] | oxygen reduction | slow-fast kinetics | [O.sub.2] channel doi/ 10.1073/pnas.1008603107

Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly, [cbb.sub.3]-type cytochrome c oxidases ([cbb.sub.3]-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for [cbb.sub.3]-Cox stability. Biogenesis of [cbb.sub.3]-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active [cbb.sub.3]-Cox. Most bacteria expressing [cbb.sub.3]-Cox also contain the ccoGHIS genes, which encode putative [cbb.sub.3]-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in [cbb.sub.3]-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out--C-terminus-in ([N.sub.out]-[C.sub.in]) topology. In its absence, neither the fully assembled [cbb.sub.3]-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of [cbb.sub.3]-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active [cbb.sub.3]-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active [cbb.sub.3]-Cox. Surprisingly, CcoH does not seem to interact only transiently with the [cbb.sub.3]-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the [cbb.sub.3]-Cox than an assembly factor per se. doi: 10.1128/JB.00988-10

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of [O.sub.2] to [H.sub.2]O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a 'gate,' which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation. conformational change | mass spectrometry | hydrogen/deuterium exchange

Structural and chemical changes in the [P.sub.M) intermediate of Paracoccus denitrificans cytochrome c oxidase are investigated using infrared spectroscopy with labeled tyrosines and histidine. The results suggest a structural alteration in P(sub M) that is centered on the His276-Pro277-Glu278-Val279-Tyr280 pentapeptide formed by the His-Tyr covalent linkage to mediate the perturbation of the infrared band of the protonated Glu278 headgroup.

Several studies and experiments are conducted to show that the major proton-transfer pathway in the buried site of cytochrome c oxidase (CcO) is the D-pathway that begins in the absence of subunit III on the inner protein surface. The results demonstrate that the subunit III makes a barrier for the protons and hence prevents them from directly accessing the internal depths of the D-pathway, though it also helps in increasing the rate at which the protons are transferred in the pathway.

Purified bovine heart cytochrome c oxidase (CcO) was extracted from aqueous solution into hexane in the presence of phospholipids and calcium ions. The enhanced rate of binding of cyanide to heme a(sub 3) was described by the destabilization of an intrinsic ligand, located at the catalytic site, that was triggered by the reduction of heme a and Cu(sub A).

An aspartic acid is introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects are examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity.

Optical spectroscopy, MCD and stopped-flow for the pH sensitivity of spectral features are used to investigate the three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a3 and Cu(sub B) are reduced and stabilized by carbon monoxide (MV.CO). Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the proteolytic residue that interacts with both hemes through a hydrogen-bonding network.

Mitochondrial (mt) sequences from cytochrome oxidase subunit I to the subunit II gene (COI, COII) in crustacean talitrid amphipods were analyzed. The COI-NC-COII organizations and preliminary results, based on both COI-COII nucleotide and amino-acid sequence indicated monopoly of these talitrid taxa.

The continuum electrostatics program multiconformation continuum (MCCE) is used to compare the electrochemical behavior of heme a in wild-type and S44D cytochrome c oxidase (CcO). The findings provide insight into the pH dependent stabilization of a heme oxidation in a model complex by a hydrogen bond to the axial His ligand.

The time-resolved reaction between fully reduced cytochrome [ba.sub.3] from Thermus thermophilus and [O.sub.2] in variants where Thr-312 and Thr-315 are modified is examined. The results have shown that proton uptake is inhibited in the two variants, which has suggested involvement of T312 and T315 in the transfer of protons to the achieve site during [O.sub.2] reduction in the [ba.sub.3] oxidase.

Site-directed mutagenesis was used to identify components of Cu(I) chaperone (Cox11) that are not required for copper binding but are absolutely required for the assembly of [Cu.sub.B], including conserved Cys-35 and Lys-123. The findings for the properties of Lys-123 and the apparent specificity of Cox11 indicated the significant role of Lys-123 in the interaction of Cox11 with its target.

The characteristics of additional mutations near the entrance region of the protein (N139, N207, N121, and S142) are determined to understand the decoupling mechanism in the D-channel of the [aa.sub.3]-type cytochrome c oxidase from Rhodobacter sphaeroides. The mechanism of decoupling could most likely be attributed to the slowing of the rate of proton delivery below a threshold, required for protonation of the putative proton loading site.

The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides which results in a ~15-fold decrease in the turnover number and a loss of proton pumping is described. The results help to resolve the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme with the net electrogenicity of the F to O transition coupled to transmembrane transfer of two charges per electron.

The molecular design of the individual components required for the direct coupling of the [O.sub.2]-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803 is described. The designed strategy facilitates the quantitative characterization of the structure, composition, and light-mediated [H.sub.2] evolution activity of the respective protein monolayer by spectroelectrochemical methods.

A study was conducted to examine the role of copper in regulation of ligand binding in [Cu.sub.B]Mb, a mutant of sperm whale myoglobin as a model for cytochrome c oxidase (CcO). The data demonstrated that the [Cu.sub.B]Mb/[Cu.sup.+] complex reproduces essential structural and kinetic features of CcO very much similar to the complex in Thermus thermophilus CcO (TtCcO) than to bovine CcO (bCcO).

The reaction chemistry and redox protein property of BSco, the homologue from Bacillus subtilis was explored to gain insight into critical role of H135 residue towards the assembly of cytochrome oxidases and possible regulation of the transfer of copper into the [Cu.sub.A] binuclear site. The results indicated that the Cu(II)-H135 interaction might be perturbed by the interaction between Sco and its protein partner(s) to induce a sensitive redox activity to the protein.

A study was conducted to examine the stability of purified, detergent-solubilized, cytochrome c oxidase (CcO) using two chemical denaturants, urea and guanidinium chloride (GdmCl). Findings reveal that GdmCl-induced dissociation of subunits occurs at lower concentrations and with a narrower concentration range.

The x-ray crystal structures of the reduced (at 2.15 [Angstrom resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides was compared to understand the displacement mechanism of heme [a.sub.3] which involved both the porphyrin ring and the hydroxyl farnesyl tail. All high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase showed a water molecule in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path.

The mechanisms involved in the formation of a covalent link between the distal tryptophan (Trp51) and the heme in cytochrome c peroxidase (CcP) are discussed. The properties of the radical thus formed are shown to be highly dependent on the structure of the compound I intermediate.

The functional model of cytochrome c oxidase (CcO) has catalyzed the four-electron reduction of [O.sub.2] by using the actual biological reductant Cytochrome c (Cytc) in the homogeneous solution. Single and steady-state turnover kinetics studies have shown that [O.sub.2] binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are fast.

The molecular model and computational protocol used to perform microscopic p[K.sub.a] calculations for the key residue, Glu286, in cytochrome c oxidase (CcO) was analyzed as stringent tests for the molecular model and computational protocol.The results confirmed the significant degree of internal consistency and reproducibility that support the effectiveness of the computational framework

A study related to the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein is presented. The results indicated that the membrane plays a significant role to accelerate proton uptake by the membrane-bound proton transporter.

The anion binding behavior of the Mg(Mn) site in cytochrome c oxidase from Rhodobacter sphaeroide is examined to understand its role in proton pumping at Mg(Mn) center when [Cu.sub.A] is oxidized. The results for the aqueous ligands of Mn(II) probed with deuterium, cyanide, and azide under reduced and oxidized conditions showed that cyanide binds to the Mn(II) ion of the [Cu.sub.A]-Mg(Mn) site when [Cu.sub.A] is reduced but not when [Cu.sub.A] is oxidized.

A mechanism is described for proton pumping by the B-type cytochrome c oxidases, where one proton is pumped in conjunction with the weakly exergonic, two-electron reduction of Fe-bound [O.sub.2] to the Fe-Cu bridging peroxodianion and three protons are pumped in conjunction with the highly exergonic, two-electron reduction of Fe(III)-[super -]O-[super -]O-Cu(II) to form water and the active oxidized enzyme. This method is based on the active-site structure of cytochrome [ba.sub.3] from Thermus thermophilus, which is required for coupled [O.sub.2] reduction and proton pumping when appropriate gates are in place.

The serine 44 residue in Rhodobacter sphaeroides eukaryotic cytochrome c oxidase (Rs CcO) was mutated to a glycine to create a bovine-like mutant form, S44G, to test the possible effect of hydrogen bonding on the histidine ligand of heme a. The hydrogen-bonding partner of the histidine ligand of heme a is found to be determinant of the EPR spectral differences between bovine and bacterial CcO and the heme a redox potential could be altered by a nearby carboxyl group whose protonation leads to a proton-coupled electron transfer process.

The first resonance Raman studies of the mixed-valence and fully reduced forms of Paracoccus pantrotrophus bacterial cytochorme c peroxidase are reported. The results from Raman spectra provide insight into the effects of reduction and calcium ion binding on the coordination and spin states of the hemes of the enzymes.

An experiment was designed to investigate electrogenic reactions in the structural variants of the cytochrome c oxidase, where in the residues in the second, K proton pathway of cytochrome c oxidase, were modified. The results indicated that the electrogenic reaction associated with the electron transfer to the catalytic sites originates as a result of charge transfer within the K pathway.

The cytochrome c oxidase (CcO) from R. sphaeroides was used to study the effect of the packing density of proteins on their activity. The results revealed that the redox activity of the enzyme is preserved in the biomimetic structure at a moderate surface coverage in which a continuous lipid bilayer is present and the protein are not forced to aggregate.

The studies of slow pH dependent conformation equilibrium between a valence-delocalized and a valence-trapped species of the dinuclear [Cu.sub.A] domain of cytochrome c oxidase from Thermus thermophilus are reported. The results provide insight into the exchange dynamics and origin of the thermostability of the proteins.

The effects of the E286H mutation on the activity and structure of cytochrome c oxidase (CytcO) are studies by using amide hydrogen-deuterium exchange mass spectrometry. The results have shown that proton uptake/transfer through the D-pathway is linked to the gating region and exit channel for pumped protons and that the mutation of E286 has perturbed the redox-based linkage.

A new photoactive binuclear ruthenium complex is utilized to study the single-electron reduction of the pulsed [O.sub.H] state of detergent-solubilized cytochrome oxidase. The findings prove that [Cu.sub.B] is the initial electron acceptor in the process.

A study was conducted to assess the extent of genetic structure displayed by Nassarius reticulatus population along South European Atlantic coastline. It was seen that a significant trend of genetic isolation by distance showed restricted dispersal and gene flow.