A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S–9S poly(A)-containing RNA from rat liver was constructed in Image . Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3′-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.

Oxygen reduction and proton translocation by cytochrome c oxidase

Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.

Reversible electrochemistry of cytochrome c on recompressed, binderless exfoliated graphite electrodes

The direct electrochemistry of cytochrome c (cyt-c) has been investigated on exfoliated graphite (EG) electrodes. The as-polished and roughened (using SiC emery sheet) EG surfaces are inactive for the direct electron transfer. However, when the EG electrode was sonicated before the experiment, a pair of redox waves were obtained for freely diffusing cyt-c in the solution phase. The formal potential was found to be 0.01 V (vs. SCE) in 0.1 M phosphate buffer at a pH of 7.1. The electrochemical response for the adsorbed cyt-c on sonicated EG electrodes, which is shown to have carbonyl functional groups on its surface, shows nearly reversible voltammograms in the same electrolyte. However, the formal potential in the adsorbed state is more negative than that observed for the solution phase cyt-c. A structure based on an open heme conformation proposed by Hildebrandt and Stockburger is probably present on the EG surface. It is suggested that the electrochemistry at the EG electrode is essentially governed by favourable electrostatic interactions.

Brown adipose tissue mitochondria are cytochrome c — subsaturated

The oxidative activity of mitochondria freshly isolated from brown adipose tissue of rats was stimulated two-fold on the addition of small concentrations of exogenous cytochrome c to the reaction medium. Loss of membrane-bound cytochrome c did not occur during isolation of mitochondria. Estimation of the high-affinity binding sites on the organelle membrane indicated that less than a third of these sites remained saturated with cytochrome c. The pigment is thus shown to be a functionally limiting electron transport component in brown adipose tissue.

Effect of beta-lactamase-catalyzed hydrolysis of cephalosporins on peroxynitrite-mediated nitration of serum albumin and cytochrome c

The hydrolysis of beta-lactam antibiotics by beta-lactamases is one of the major bacterial defense systems. These enzymes generally hydrolyze a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. In this paper, the effect of cephalosporins-based antibiotics on the peroxynitrite-mediated nitration of protein tyrosine is described. Although some of the antibiotics have weak inhibitory effect on the nitration reactions in the absence of beta-lactamase, they exhibit very strong inhibition in the presence of beta-lactamase. This is due to the elimination of heterocyclic thiol/thione moieties from cephalosporins by beta-lactamase-mediated hydrolysis. After the elimination, the thiols/thiones effectively scavenge peroxynitrite, leading to the inhibition of the nitration reactions.

Electron transfer in cytochrome c oxidase : the rate of electron equillibration between cytochrome a and copper A

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by means of perturbed equilibrium techniques. We have prepared a three electron reduced, CO inhibited form of the enzyme in which cytochrome a and copper A are partially reduced an in intramolecular redox equilibrium. When these samples were photolyzed using a nitrogen laser (0.6 µs, 1.0 mJ pulses) changes in absorbance at 598 nm and 830 nm were observed which are consistent with a fast electron from cytochrome a to copper A. The absorbance changes at 598 nm have an apparent rate of 17,200 ± 1,700 s^(-1) (1σ), at pH 7.0 and 25.5 °C. These changes were not observed in either the CO mixed valence or CO inhibited fully reduced forms of the enzyme. The rate is fastest at about pH 8.0, and falls off in either direction, and there is a small, but clear temperature dependence. The process was also observed in the cytochrome c -- cytochrome c oxidase high affinity complex.

This rate is far faster than any rate measured or inferred previously for the cytochrome a -- copper A electron equilibration, but the interpretation of these results is hampered by the fact that the relaxation could only be followed during the time before CO became rebound to the oxygen binding site. The meaning of our our measured rate is discussed, along with other reported rates for this process. In addition, a temperature-jump experiment on the same system is discussed.

We have also prepared a partially reduced, cyanide inhibited form of the enzyme in which cytochrome a, copper A and copper B are partially reduced and in redox equilibrium. Warming these samples produced absorbance changes at 605 nm which indicate that cytochrome a was becoming more oxidized, but there were no parallel changes in absorbance at 830 nm as would be expected if copper A was becoming reduced. We concluded that electrons were being redistributed from cytochrome a to copper B. The kinetics of the absorbance changes at 605 nm were investigated by temperature-jump methods. Although a rate could not be resolved, we concluded that the process must occur with an (apparent) rate larger than 10,000 s^(-1).

During the course of the temperature-jump experiments, we also found that non-redox related, temperature dependent absorbance changes in fully reduced CO inhibited cytochrome c oxidase, and in the cyanide mixed valence enzyme, took place with an (apparent) rate faster that 30,000 s^(-1).

Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNA(ser) is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

Water-soluble phosphate-functionalized polyfluorene as fluorescence biosensors toward cytochrome c

An anionic water-soluble polyfluorene derivative, poly(9,9-bis(6'-phosphatehexyl)fluorene-alt-1,4-phenylene) sodium salt (PFHPNa), was synthesized by Suzuki coupling reaction in DMF/water. Polymer PFHPNa was well soluble in water with a strong blue fluorescence emission. Effect of the side chain length on fluorescence sensory properties was studied by comparing quenching efficiencies toward different quenchers of PFHPNa with a reported polymer poly(9,9-bis(3'-phosphatepropyl)fluorene-alt-1,4-phenylene) sodium salt (PFPPNa), which have different side chains in length. For small molecular quenchers (methylviologen, MV2+) and meso-5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4), polymer PFHPNa had lower sensitivity due to the much longer side chain length. The positively charged metalloprotein cytochrome c could quench fluorescence of conjugated polymers via energy transfer and electron transfer.

Surface plasmon resonance and electrochemistry characterization of layer-by-layer self-assembled DNA and Zr4+ thin films, and their interaction with cytochrome c

Through electrostatic layer-by-layer (LbL) assembly, negatively charged calf thymus double stranded DNA (CTds-DNA), and positively charged Zr4+ ions were alternately deposited on gold substrate modified with chemisorbed cysteamine. Thus-prepared three-dimensional DNA networks were characterized by surface plasmon resonance (SPR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and infrared reflection-absorption spectroscopy (IR-RAS). SPR spectroscopy indicates that the effective thickness of DNA monolayer in the (DNA/Zr4+), bilayer was 1.5 +/- 0.1 nm, which corresponds to the surface coverage of 79% of its full packed monolayer. At the same time, a linear increase of film thickness with increasing number of layers was also confirmed by SPR characterizations. The data of XPS and IR-RAS show that Zr4+ ions interact with both the phosphate groups and nitrogenous bases of DNA and load into the framework of DNA. Furthermore, the interactions between this composite film and heme protein cytochrome c (Cyt c) were investigated by SPR spectroscopy and electrochemistry.

Surface plasmon resonance and electrochemistry characterization of layer-by-layer self-assembled DNA and Zr4+ thin films, and their interaction with cytochrome c

Through electrostatic layer-by-layer (LbL) assembly, negatively charged calf thymus double stranded DNA (CTds-DNA), and positively charged Zr4+ ions were alternately deposited on gold substrate modified with chemisorbed cysteamine. Thus-prepared three-dimensional DNA networks were characterized by surface plasmon resonance (SPR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and infrared reflection-absorption spectroscopy (IR-RAS). SPR spectroscopy indicates that the effective thickness of DNA monolayer in the (DNA/Zr4+), bilayer was 1.5 +/- 0.1 nm, which corresponds to the surface coverage of 79% of its full packed monolayer. At the same time, a linear increase of film thickness with increasing number of layers was also confirmed by SPR characterizations. The data of XPS and IR-RAS show that Zr4+ ions interact with both the phosphate groups and nitrogenous bases of DNA and load into the framework of DNA. Furthermore, the interactions between this composite film and heme protein cytochrome c (Cyt c) were investigated by SPR spectroscopy and electrochemistry. Compared with the adsorption of Cyt c on DNA monolayer, this composite multilayer film can obviously enhance the amount of immobilized Cyt c confirmed by SPR reflectivity-incident angle (R-theta) curves.

Direct electrochemistry behavior of Cytochrome c on silicon dioxide nanoparticles-modified electrode

A newfangled direct electrochemistry behavior of Cytochrome c (Cyt c) was found on glassy carbon (GC) electrode modified with the silicon dioxide (SiO2) nanoparticles by physical adsorption. A pair of stable and well-defined redox peaks of Cyt c ' quasi-reversible electrochemical reaction were obtained with a heterogeneous electron transfer rate constant of 1.66 x 10(-3) cm/s and a formal potential of 0.069 V (vs. Ag/AgCl) (0.263 V versus NHE) in 0.1 mol/L pH 6.8 PBS. Both the size and the amount of SiO2 nanoparticles could influence the electron transfer between Cyt c and the electrode. Electrostatic interaction which is between the negative nanoparticle surface and positively charged amino acid residues on the Cyt c surface is of importance for the stability and reproducibility toward the direct electron transfer of Cyt c. It is suggested that the modification of SiO2 nanoparticles proposes a novel approach to realize the direct electrochemistry of proteins.

Direct electron transfer between cytochrome c and a gold nanoparticles modified electrode

Quasi-reversible and direct electrochemistry of cytochrome c (cyt. c) has been obtained at a novel electrochemical interface constructed by self-assembling gold nanoparticles (GNPs) onto a three-dimensional silica gel network, without polishing or any modification of the surface. A cleaned gold electrode was first immersed in a hydrolyzed sol of the precursor (3-mercaptopropyl)-trimethoxysilane to assemble three-dimensional silica gel, then the GNPs were chemisorbed onto the thiol groups of the sol-gel network and modified the kinetic barrier of this self-assembled silicate film. Cyclic voltammetry and AC impendance spectroscopy were performed to evaluate electrochemical properties of the as prepared interface. These nanoparticle inhibits the adsorption of cyt. c onto bare electrode and acts as a bridge of electron transfer between protein and electrode.

Surface-enhanced resonance Raman spectroscopy and spectroscopy study of redox-induced conformational equilibrium of cytochrome c adsorbed on DNA-modified metal electrode

The redox-induced conformational equilibrium of cytochrome c (cyt c) adsorbed on DNA-modified metal electrode and the interaction mechanism of DNA with cyt c have been studied by electrochemical, spectroscopic and spectroelectrochemical techniques. The results indicate that the external electric field induces potential-dependent coordination equilibrium of the adsorbed cyt c between its oxidized state (with native six-coordinate low-spin and non-native five-coordinate high-spin heme configuration) and its reduced state (with native six-coordinate low-spin heme configuration) on DNA-modified metal electrode. The strong interactions between DNA and cyt c induce the self-aggregation of cyt c adsorbed on DNA. The orientational distribution of cyt c adsorbed on DNA-modified metal electrode is potential-dependent, which results in the deviation from an ideal Nernstian behavior of the adsorbed cyt c at high electrode potentials. The electric-field-induced increase in the activation barrier of proton-transfer steps attributed to the rearrangement of the hydrogen bond network and the self-aggregation of cyt c upon adsorption on DNA-modified electrode strongly decrease the interfacial electron transfer rate.