Crystallographic cyanide-probing for cytochrome c oxidase reveals structural bases suggesting that a putative proton transfer H-pathway pumps protons.

Cytochrome c oxidase (CcO) reduces O in the O-reduction site by sequential four-electron donations through the low-potential metal sites (Cu and Fe). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp, Arg, Glu, the hydroxyfarnesyl ethyl group of heme a, and Ser, respectively. These sites interact with the putative proton-pumping H-pathway.

Solid-Phase Synthesis of Glycosyl Phosphate Repeating Units via Glycosyl Boranophosphates as Stable Intermediates.

Solid-phase synthesis of glycosyl phosphate repeating units was investigated using glycosyl boranophosphates as stable precursors. The stable nature of glycosyl boranophosphate enables the elongation of a saccharide chain without remarkable decomposition. After deprotection of the boranophosphotriester linkages to boranophosphodiesters, the intersugar linkages were converted to the phosphate counterparts quantitatively using an oxaziridine derivative.

Calcium-bound structure of bovine cytochrome c oxidase.

The crystal structure of bovine cytochrome c oxidase (CcO) shows a sodium ion (Na) bound to the surface of subunit I. Changes in the absorption spectrum of heme a caused by calcium ions (Ca) are detected as small red shifts, and inhibition of enzymatic activity under low turnover conditions is observed by addition of Ca in a competitive manner with Na. In this study, we determined the crystal structure of Ca-bound bovine CcO in the oxidized and reduced states at 1.

Biochemical and crystallographic studies of monomeric and dimeric bovine cytochrome oxidase.

Cytochrome oxidase (CcO), a terminal oxidase in the respiratory chain, catalyzes the reduction of O to water coupled with the proton pump across the membrane. Mitochondrial CcO exists in monomeric and dimeric forms, and as a monomer as part of the respiratory supercomplex, although the enzymatic reaction proceeds in the CcO monomer. Recent biochemical and crystallographic studies of monomeric and dimeric CcOs have revealed functional and structural differences among them.

Solid-Phase Stereocontrolled Synthesis of Oligomeric P-Modified Glycosyl Phosphate Derivatives Using the Oxazaphospholidine Method.

Glycosyl phosphate repeating units can be found in the glycoconjugates of some bacteria and protozoa parasites. These structures and their P-modified analogs are attractive synthetic targets as antimicrobial, antiparasitic, and vaccine agents. However, P-modified glycosyl phosphates exist in different diastereomeric forms due to the chiral phosphorus atoms, whose configuration would highly affect their physiochemical and biochemical properties.

The 1.3-Å resolution structure of bovine cytochrome oxidase suggests a dimerization mechanism.

Cytochrome oxidase (CcO) in the respiratory chain catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme and proton pumping across the membrane. Although the functional unit of CcO is monomeric, mitochondrial CcO forms a monomer and a dimer, as well as a supercomplex with respiratory complexes I and III. A recent study showed that dimeric CcO has lower activity than monomeric CcO and proposed that dimeric CcO is a standby form for enzymatic activation in the mitochondrial membrane.

The active form of quinol-dependent nitric oxide reductase from is a dimer.

is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from (qNOR) reported here at 3.

Monomeric structure of an active form of bovine cytochrome oxidase.

Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important.

X-ray structural analyses of azide-bound cytochrome oxidases reveal that the H-pathway is critically important for the proton-pumping activity.

Cytochrome oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme (Fe ), for reduction of O at another iron site, heme (Fe ). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway.

Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme.

Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to NO and HO. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic.

The Mg2+-containing Water Cluster of Mammalian Cytochrome c Oxidase Collects Four Pumping Proton Equivalents in Each Catalytic Cycle.

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by HO through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O bound to heme a To block backward proton movement, the water channel remains closed after O binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O binding.

Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL.

We report a method of femtosecond crystallography for solving radiation damage-free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage-free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

Reaction mechanism of mammalian mitochondrial cytochrome c oxidase.

Cytochrome c oxidase (COX) is the terminal oxidase of the mitochondrial respiratory system. This enzyme reduces molecular oxygen (O(2)) to water in a reaction coupled with the pumping of protons across the mitochondrial inner membrane. Progress in investigating the reaction mechanism of this enzyme has been limited by the resolution of its X-ray structure.

Structural studies on bovine heart cytochrome c oxidase.

Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8Å. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (Cu(A), heme a (Fe(a)), Cu(B), heme a(3) (Fe(a3))) and 3 redox-inactive metal sites (Mg(2+), Zn(2+) and Na(+)).

Proton-pumping mechanism of cytochrome C oxidase.

Cytochrome c oxidase (CcO), as the terminal oxidase of cellular respiration, coupled with a proton-pumping process, reduces molecular oxygen (O(2)) to water. This intriguing and highly organized chemical process represents one of the most critical aspects of cellular respiration. It employs transition metals (Fe and Cu) at the O(2) reduction site and has been considered one of the most challenging research subjects in life science.

X-ray structure of the NO-bound Cu(B) in bovine cytochrome c oxidase.

The X-ray crystallographic structure of nitric oxide-treated bovine heart cytochrome c oxidase (CcO) in the fully reduced state has been determined at 50 K under light illumination. In this structure, nitric oxide (NO) is bound to the CcO oxygen-reduction site, which consists of haem and a Cu atom (the haem a(3)-Cu(B) site). Electron density for the NO molecule was observed close to Cu(B).