Cytochrome A3 Research Articles

Light wavelengths that induce oxidation of oxymyoglobin in meat

Light wavelengths that induce meat discoloration and the photoreceptors in the meat were studied. We investigated the effects of the light wavelength on the oxidation rate of myoglobin (Mb) by exposing Mb extracts or model solutions containing Mb to light at specific wavelengths with a bandwidth of 5 nm using a fluorescence spectrophotometer. The wavelengths examined comprised 385, 415, 445, 460, 490, 525, 555, 580, 605, 630,660, and 750 nm. In the Mb extracts, Mb oxidation was induced through exposure to the light at 445 and 580–605 nm; Mb was insensitive to light at 445 nm. Mitochondria, containing cytochrome a and cytochrome a3 with absorption peaks at 448 and 600 nm, and riboflavin with fluorescence at 450 nm were studied as 445 nm receptors. Mitochondria significantly oxidized Mb via cytochrome c oxidation through complex IV activity; however, no 445 nm-specific photo sensitivity effects were observed. In contrast, riboflavin increased the Mb oxidation rate induced via exposure to the light at 450 nm in a concentration-dependent manner (minimum concentration: 38.4 μg L−1). While native mitochondria did not show 445 nm-specific photosensitivity effects on Mb, supernatants of heated mitochondria conferred 445 nm-wavelength sensitivity to Mb. Riboflavin concentration in this supernatant was 182 ± 60 μg L−1. The Mb photosensitivity spectrum with 473 μg L−1 riboflavin had two peaks at 445 nm and 580 nm, which were similar to those of Mb extract. These results suggest that mitochondrial damage affects the meat discoloration through the release of cytochrome c and riboflavin.

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a3 (Fea3) and copper (CuB). Here, the influence of the ligation at the oxidized Fea33+–CuB2+ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (Em(a)) for the cyanide (the low-spin Fea33+) and the formate-ligated CcO (the high-spin Fea33+). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of Em(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fea3+ between His–Fea3+–His and His–Fea3+–OH− occurs only at pH above 9.5 with the estimated pK >11.0.

Review on Cyanide Poisoning in Ruminants

Cyanide is one of the most potent and rapidly acting inorganic poisons and it can cause toxicity in animals, principally in ruminants. Although cyanides are released to environment in various forms, the natural source of cyanide ion from plants that contain enough cyanogenic glycosides are the most important cause of hydrogen cyanide poisoning in ruminants. The objective of this paper is to review available information on cyanide poisoning in ruminants and to highlight its diagnosis, prevention and treatment options. The cyanide toxicity in ruminants is affected primarily by lethal dose, plant, environment, and animal factors. Ruminants are more susceptible to prussic acid poisoning than other domestic animals, due to more efficient hydrolysis of the cyanogenic glycosides in the rumen. The mechanism of cyanide intoxication involves, inhibition of oxidative metabolism and oxygen utilization of cells, by binding with heme form of cytochrome a3 (cytochrome oxidase) which is last enzyme in the respiratory electron transport chain. As result of this process, affected ruminant ’ s shows signs of acute cyanide intoxication mainly the reflection of cellular hypoxia including hyperventilation, decreased blood pressure, hypoxemia-induced convulsions, coma, shock, respiratory failure and death within few minutes to hours. Nevertheless, in most cases the affected animals die due to rapidly acting nature of the toxin; ruminants can be treated immediately with specific antidote. Combination of sodium nitrite and sodium thiosulfate is commonly dosed orally as antidote of choice. The use of appropriate prevention measures can reduce the risk from potentially toxic forages. The rapid administration of sodium thiosulphate alone or in combination with sodium nitrite intravenously (IV) to early noticed should be done to reduce losses of livestock. Keywords : Cyanide, ruminants, toxicity, hydrogen cyanide; DOI : 10.7176/JBAH/9-6-01 Publication date :March 31 st 2019

Response of Heme Symmetry to the Redox State of Bovine Cytochrome c Oxidase.

Second-derivative absorption spectroscopy was employed to monitor the response of effective symmetry of cytochromes a and a3 to the redox and ligation states of bovine cytochrome c oxidase (CcO). The Soret band π → π* electronic transitions were used to display the changes in symmetry of these chromophores induced by the reduction of CcO inhibited by the exogenous ligands and during catalytic turnover. The second derivative of the difference absorption spectra revealed only a single Soret band for the oxidized cytochromes a and a3 and cyanide-ligated oxidized cytochrome a3. In contrast, two absorption bands were resolved in ferrous cytochrome a and ferrous cytochrome a3 ligated with cyanide. A transition from one-band spectrum to two-band spectrum indicates the lowering of symmetry of these hemes due to the alteration of their immediate surroundings. It is suggested that the changes in polarity occurring in the vicinity of these cofactors are main reason for the split of the Soret band of both ferrous cytochrome a and cyanide-bound ferrous cytochrome a3.

Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell

Membraneless microbial fuel cell (MFC) employing new microbial catalyst formed as yeast cultivated from Saccharomyces cerevisiae and carbon nanotube (yeast/CNT) is suggested. To analyze its catalytic activity and performance and stability of MFC, several characterizations are performed. According to the characterizations, the catalyst shows excellent catalytic activities by facile transfer of electrons via reactions of NAD, FAD, cytochrome c and cytochrome a3, while it induces high maximum power density (MPD) (344mW·m−2). It implies that adoption of yeast induces increases in catalytic activity and MFC performance. Furthermore, MPD is maintained to 86% of initial value even after eight days, showing excellent MFC stability.

Revisiting individual absorption spectra of reduced hemes a and a3 in bovine-heart cytochrome c oxidase

Revisiting individual absorption spectra of reduced hemes a and a3 in bovine-heart cytochrome c oxidase

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the CuA center of cytochrome c oxidase

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the CuA center contained within subunit II of the oxidase complex. The CuA center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a3 and CuB. CuA consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of CuA present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in CuA assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-CuA site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Enhancing T3 and cAMP responsive gene participation in the thermogenic regulation of fuel oxidation pathways

We sought to identify glycolysis, glycogenolysis, lipolysis, Krebs cycle, respiratory chain, and oxidative phosphorylation enzymes simultaneously regulated by T3 and cAMP. We performed in silico analysis of 56 promoters to search for cis-cAMP (CREB) and cis-thyroid (TRE) response elements, considering UCP1, SERCA2 and glyceraldehyde 3-phosphate dehydrogenase as reference. Only regulatory regions with prior in vitro validation were selected. 29/56 enzymes presented potential TREs in their regulatory sequence, and some scored over 0.80 (better predictive value 1): citrate synthase, phosphoglucose isomerase, succinate dehydrogenases A/C, UCP3, UCP2, UCP4, UCP5, phosphoglycerate mutase, glyceraldehyde 3-P dehydrogenase, glucokinase, malate dehydrogenase, acyl-CoA transferase (thiolase), cytochrome a3, and lactate dehydrogenase. Moreover, some enzymes have not yet been described in the literature as genomically regulated by T3. Our results point to other enzymes which may possibly be regulated by T3 and CREB, and speculate their joint roles in contributing to the optimal thermogenic acclimation.

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase

The propionate groups of heme a and a3 in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a3, as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H2O–D2O isotope substitution experiments combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).

PFA: Program for the Quantitative Assessment of Cell Metabolism by Spectral Data Analysis

Assessment of mitochondrial oxidative metabolism has wide-ranging importance, from pharmacokinetic analysis to studies in cell viability and apoptosis. Here we present the Perfusion File Analyzer (PFA) application for the real-time analysis of spectral data to measure cytochrome c reduction, cytochrome a3 reduction, and other parameters important to cellular metabolism, which are collected during tissue perfusion experiments. Our current efforts are focused on quantitating changes in mitochondrial function by normalizing baseline drift of spectral data while addressing two major challenges: (1) a lack of real-time feedback from the system when aiming is compromised, and (2) an inability to adjust calculated data in the event of spectral shift. PFA has been developed to address these issues, and is currently used for quality assessment of human islets prior to clinical transplantation.

S13.37 A sperm whale myoglobin as protein model of cytochrome a3: The role of heme propionates

S13.37 A sperm whale myoglobin as protein model of cytochrome a3: The role of heme propionates

Quantifying the role of oxygen pressure in tissue function

it has been suggested that oxygen had reached significant levels in the earth's oceans as early as 2.5 billion years ago and has rapidly increased to near current levels about 50 million years later ([6][1]). The appearance of oxygen dramatically changed the chemical composition of the oceans and

Rat carotid body chemosensory discharge and glomus cell HIF-1α expression in vitro: regulation by a common oxygen sensor

Addition of Pco ( approximately 350 Torr) to a normoxic medium (Po(2) of approximately 130 Torr) was used to investigate the relationship between carotid body (CB) sensory discharge and expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) in glomus cells. Afferent electrical activity measured for in vitro-perfused rat CB increased rapidly (1-2 s) with addition of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr), and this increase was fully reversed by white light. At submaximal light intensities, the extent of reversal was much greater for monochromatic light at 430 and 590 nm than for light at 450, 550, and 610 nm. This wavelength dependence is consistent with the action spectrum of the CO compound of mitochondrial cytochrome a(3). Interestingly, when isolated glomus cells cultured for 45 min in the presence of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr) in the dark, the levels of HIF-1 alpha, which turn over slowly (many minutes), increased. This increase was not observed if the cells were illuminated with white light during the incubation. Monochromatic light at 430- and 590-nm light was much more effective than that at 450, 550, and 610 nm in blocking the CO-induced increase in HIF-1 alpha, as was the case for chemoreceptor discharge. Although the changes in HIF-1 alpha take minutes and those for CB neural activity occur in 1-2 s, the similar responses to CO and light suggest that the oxygen sensor is the same (mitochondrial cytochrome a(3)).

Evidence for a common oxygen sensor for chemosensory discharge and HIF1a expression in glomus cells in rat carotid body

Addition of a partial pressure of carbon monoxide (PCO ~ 350 Torr) to a normoxic medium (PO2 ~ 130 Torr), was used to investigate the relationship between carotid body (CB)sensory discharge and expression of hypoxia-inducible factor-1 (HIF-1) in glomus cells. Afferent electrical activity measured for in vitro perfused rat CB increased rapidly (2–3 seconds)with addition of high CO (PCO ~ 350 Torr; Po2 ~ 130 Torr) and this increase was fully reversedby white light. At submaximal light intensities, the extent of reversal was much greater formonochromatic light at 430 nm and 590 nm than for light at 450 nm, 550 nm and 610 nm. This wavelength dependence is consistent with the action spectrum of the CO compound of mitochondrial cytochrome a3. Interestingly, when isolated glomus cells cultured for 45 min inthe presence of high CO (PCO ~ 350 Torr; Po2 ~ 130 Torr) in the dark, the levels of HIF-1, which turns over slowly (many minutes), increased. This increase was not observed if the cells were illuminated with white light during the incubation. Monochromatic light at 430 nm and 590 nm light was much more effective than that at 450, 550, and 610 nm in blocking the CO induced increase in HIF-1, as was observed for the CO induced increase in chemoreceptor discharge. This similarity indicates they are responding to the same oxygen sensor.

Immediate and long-term responses of the carotid body to high altitude.

High altitude and the decreased environmental oxygen pressure have both immediate and chronic effects on the carotid body. An immediate effect is to limit the oxygen available for mitochondrial oxidative phosphorylation, and this leads to increased activity on the afferent nerves leading to the brain. In the isolated carotid body preparation, the afferent nerve activity depends on the ratio of carbon monoxide (CO), an inhibitor of respiratory chain function, to oxygen. The CO-induced increase in afferent neural activity is reversed by light, and the wavelength dependence of this reversal shows that the site of CO (and therefore oxygen) interaction is cytochrome a3 of the mitochondrial respiratory chain. Thus, primary sensing of ambient oxygen pressure is through the oxygen dependence of mitochondrial oxidative phosphorylation. The conductance of ion channels in the cellular membranes may also be sensitive to oxygen pressure and, through this, modulate the sensitivity to oxygen pressure. Longer-term exposure to high altitude results in progressive changes in the carotid body that involve several mechanisms, including cellular energy metabolism and hypoxia inducible factor-1alpha (HIF-1alpha). These changes begin within minutes of exposure, but progress such that chronic exposure results in morphological and biochemical alterations in the carotid body, including enlarged cells, increased catecholamine levels, altered cellular appearance, and others. In the chronically adapted carotid body, responses to acute changes in oxygen pressure are enhanced. The adaptive changes due to chronic hypoxia are largely reversed upon return to lower altitudes.

Structure of valence band in cluster model for cytochrome-c-oxidase active centers

The electron structure of cluster model for active centers of cytochrome-c-oxidase has been calculated by DFT (PBE) method in 6-31G basis. The cluster model was constructed on the basis of experimental PDB-structure and included 1105 hemoprotein atoms. The valence band ceiling of cytochromeoxidase active centers is shown to be formed by the atoms of carbon and nitrogen from porphyrin ring of cytochrome heme a3 and atoms of carbon and nitrogen from imidazol moieties of histidine connected with Cu atom in cytochrome a3. D-orbitals of Cu and Fe atoms from heme a3 and d-orbital of Fe atom from heme a contribute mainly to the orbital group. A conclusion is made that the catalytic activity of the structure is determined by these two types of orbitals. Cu d-orbitals of cytochrome a are substantially low in energy. It is suggested that Cu atoms of cytochrome a shift the chemical potential of d-orbitals belonging to the active center that results in their easier electron accepting and releasing. This can be a decisive factor in the electron transport process.

Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.

Unusual cytochrome a592 with low PO2 affinity correlates as putative oxygen sensor with rat carotid body chemoreceptor discharge.

Light-absorption spectra and afferent chemoreceptor discharge were simultaneously recorded on superfused rat carotid bodies (CBs) under the influence of cytochrome a3-CuB ligands (O2, CN-, CO) in order to identify the primary mitochondrial cytochrome c oxidase (CCO) oxygen sensor. Spectra could be described on the basis of weighted light-absorption spectra of cytochrome b558 of the NAD(P)H oxidase and mitochondrial cytochromes b and c, CCO, cytochrome a3, and an unusual cytochrome a peaking at 592 nm. Discharge signals were deconvoluted into phasic and tonic activity for comparing different CB responses. The spectral weight of cytochrome a592 decreased significantly starting at high PO2 (100 mm Hg) and low sodium cyanide (CN-, 10 mM) accompanied by increasing phasic peak discharge. Combined CO-hypoxia or CO-CN- application inhibited photolysis of CO-stimulated chemoreceptor discharge, revealing photometrically cytochrome a592 as central in oxygen sensing. Control spectra in tissue from sympathetic and nodose ganglia did not show any cytochrome a592 contribution. According to these results, cytochrome a592 is assumed as a unique component of CB CCO, revealing in contrast to other cytochromes an apparent low PO2 and high CN- affinity, probably due to a shortcut of electron flow within CCO between CuA and cytochrome a3-CuB.

Cytochrome oxidase sensitivity to carbon monoxide in leaves of various tall and dwarf wheat cultivars and their crosses

In order to investigate the possible role of Rht genes in the regulation of the redox condition of cytochrome a3 (cytochrome c oxidase) during steady-state respiration, wheat cultivars belonging to one of two groups – NP 710, NP 846 and NP 875 belonging to the tall group and Olesons dwarf, HD 1982 and HD 2122 of the dwarf group – and the reciprocal crosses between the varieties of these two groups were examined for carbon monoxide (CO) sensitivity in terms of the inhibition of mitochondrial electron transport. Leaves of young wheat seedlings were used. Differences in the redox state of cytochrome a3 were monitored using the in vivo aerobic assay of nitrate reduction after a 1-min exposure to CO. Dwarf cultivars possessing Rht genes responded marginally (≅10%) to CO inhibition, whereas the response of tall cultivars to CO was higher (51–70%). Since CO forms a complex only with reduced cytochrome a3, the results indicate differences in the redox state of cytochrome a3 during in situ respiration of leaves from tall and dwarf plants that are likely to be controlled by cytoplasmic factors.