Structural and functional mechanisms of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in mitochondria. It catalyzes the four-electron reduction of O to HO and harnesses the redox energy to drive unidirectional proton translocation against a proton electrochemical gradient. A great deal of research has been conducted to comprehend the molecular properties of CcO.

Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

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. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O state, and a reductive phase, in which O is reduced back to the R state.

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Cytochrome oxidase (C O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme group in their active sites, C O has a unique binuclear center (BNC) comprised of a copper atom (Cu ) and a heme iron, where O binds and is reduced to water. CO is a versatile O surrogate in ligand binding and escape reactions.

Structural basis for functional properties of cytochrome oxidase.

Cytochrome oxidase (CO) 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. The full turnover of the CO reaction involves an oxidative phase, in which the reduced enzyme () is oxidized by molecular oxygen to the metastable oxidized state, and a reductive phase, in which is reduced back to the state.

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, Cu and heme a, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand-binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome oxidase.

Cytochrome oxidase (CO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CO. It is assigned to the P-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation.

Crystal structure of CO-bound cytochrome oxidase determined by serial femtosecond X-ray crystallography at room temperature.

Cytochrome oxidase (CO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations.

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay.

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae.

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH.

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.

Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident.

Proton translocation in cytochrome c oxidase: insights from proton exchange kinetics and vibrational spectroscopy.

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain. It reduces oxygen to water and harnesses the released energy to translocate protons across the inner mitochondrial membrane. The mechanism by which the oxygen chemistry is coupled to proton translocation is not yet resolved owing to the difficulty of monitoring dynamic proton transfer events.

Redox-controlled proton gating in bovine cytochrome c oxidase.

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix.

Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg.

Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation.

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-L-arginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J.

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors.

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.

Radical formation in cytochrome c oxidase.

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50μs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes.

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli.

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3).

Electron transfer pathways in cytochrome c oxidase.

Mixed quantum mechanical/molecular mechanics calculations were used to explore the electron pathway of the terminal electron transfer enzyme, cytochrome c oxidase. This enzyme catalyzes the reduction of molecular oxygen to water in a multiple step process. Density functional calculations on the three redox centers allowed for the characterization of the electron transfer mechanism, following the sequence Cu(A)→heme a→heme a(3).

Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E.

Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated.

EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen.

The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 micros-6 ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9 GHz) and D-band (130 GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (SO2(-.)), respectively.