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PubMed1977

Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogensis from ammonia in isolated rat hepatocytes.

1. Citrate isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions, thereof, obtained by the digitonin and silicone oil fractionation technique. 2. Caculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV. 3; Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, is demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reserved. 4. It is concluded that during urogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 muM, below the Km (isocitrate) of the NAD-linked enzyme but in the range of that for the NADP-linked enzyme. 5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate. 6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.

Ammonia↗
PubMed1980

Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II.

The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes. 2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200--350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200--400 microseconds. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1. 3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q-B leads to QB-, and following the second flash, Q-B- leads to QB2-. Benzoquinone at low concentrations oxidises a residual fraction of B- which is usually reduced in the dark before the flash sequence. 4. A faster component in the decay (t u/2 approximately 140 microseconds) following the first flash titrated in over the range Eh greater than 350 mV. The binary oscillation was still apparent but delayed by one flash. 5. We discuss the relative redox potentials of the couples B/B- and B-/B2-, and the role of the component which titrates in at Eh greater than 350 mV.

Chloroplasts↗
PubMed1991

A thermodynamic model for the cooperative functional properties of the tetraheme cytochrome c3 from Desulfovibrio gigas.

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desulfovibrio gigas. This molecule displays different intrinsic redox potentials for the four hemes and during the redox titration process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A.V. (1984) Eur. J. Biochem. 141, 283-296]. This complex cooperative behaviour [Xavier, A.V. (1986) J. Inorg. Biochem. 28, 239-243] has been analyzed here using an I2H4-interaction network [Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250] coupled to a proton-linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMR redox-titration curves. The 'redox-Bohr' effect is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed.

Binding Sites↗
PubMed1995

Iron-ligand structure and iron redox property of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum: relevance to its NO reduction activity.

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements. The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S(-)-Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not. In spite of the same S(-)-Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2005

The reactions of nitrosyl complexes with cysteine.

The reaction kinetics of a set of ruthenium nitrosyl complexes, {(X)5MNO}n, containing different coligands X (polypyridines, NH3, EDTA, pz, and py) with cysteine (excess conditions), were studied by UV-vis spectrophotometry, using stopped-flow techniques, at an appropriate pH, in the range 3-10, and T = 25 degrees C. The selection of coligands afforded a redox-potential range from -0.3 to +0.5 V (vs Ag/AgCl) for the NO+/NO bound couples. Two intermediates were detected. The first one, I1, appears in the range 410-470 nm for the different complexes and is proposed to be a 1:1 adduct, with the S atom of the cysteinate nucleophile bound to the N atom of nitrosyl. The adduct formation step of I1 is an equilibrium, and the kinetic rate constants for the formation and dissociation of the corresponding adducts were determined by studying the cysteine-concentration dependence of the formation rates. The second intermediate, I2, was detected through the decay of I1, with a maximum absorbance at ca. 380 nm. From similar kinetic results and analyses, we propose that a second cysteinate adds to I1 to form I2. By plotting ln k1(RS-) and ln k2(RS-) for the first and second adduct formation steps, respectively, against the redox potentials of the NO+/NO couples, linear free energy plots are obtained, as previously observed with OH- as a nucleophile. The addition rates for both processes increase with the nitrosyl redox potentials, and this reflects a more positive charge at the electrophilic N atom. In a third step, the I2 adducts decay to form the corresponding Ru-aqua complexes, with the release of N2O and formation of cystine, implying a two-electron process for the overall nitrosyl reduction. This is in contrast with the behavior of nitroprusside ([Fe(CN)5NO]2-; NP), which always yields the one-electron reduction product, [Fe(CN)5NO]3-, either under substoichiometric or in excess-cysteine conditions.

Cysteine↗
PubMed2002

Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers.

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.

Amino Acid Sequence↗
PubMed2004

Effect of O2 exposure on perchlorate reduction by Dechlorosoma sp. KJ.

Anaerobic bioreactors have been developed to remove perchlorate from water, but backwashing and operational interruptions can expose biofilms to oxygen. While it is well known that oxygen is a preferential electron acceptor to perchlorate for perchlorate-respiring bacteria, little is known about the effect of oxygen exposure or redox potentials on perchlorate reduction. Four different dissolved oxygen scavengers were tested for their ability to quickly restore anaerobic conditions and allow perchlorate reduction by a facultative, perchlorate respiring bacterium Dechlorosoma sp. KJ. Of the four different oxygen scavengers tested (Oxyrase trade mark, L-cysteine, Na2S and FeS), only Oxyrase trade mark was able to rapidly (<30 min) scavenge dissolved oxygen and allow cell growth. There was no cell growth after addition of Na2S and FeS, and l-cysteine produced a long lag in cell growth. To investigate the effect of dissolved oxygen on perchlorate reduction, anaerobically grown cultures Dechlorosoma sp. KJ, were exposed to dissolved oxygen for various periods ranging from 1 to 32 h. Perchlorate reduction and redox potential were then measured for cells returned to an anaerobic environment containing an oxygen scavenger. It was determined that cells exposed to dissolved oxygen for more than 12h were incapable of reducing perchlorate. Cells exposed to dissolved oxygen for less than 12h quickly reduced the redox potential to negative values (-127 mV to -337 mV) and were able to reduce perchlorate or chlorite. Our results suggest that aeration during backwashing of biofilm reactors, or exposure of perchlorate-degrading cell suspensions to dissolve oxygen for less than 12h, will not be detrimental to the ability of perchlorate-degrading bacteria to use perchlorate as an electron acceptor.

Biodegradation, Environmental↗
PubMed2002

Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease.

Factors which govern transnitrosation reactions between hemoglobin (Hb) and low molecular weight thiols may define the extent to which S-nitrosated Hb (SNO-Hb) plays a role in NO in the control of blood pressure and other NO-dependent reactions. We show that exposure to S-nitrosylated cysteine (CysNO) produces equivalent levels of SNO-Hb for Hb A(0) and sickle cell Hb (Hb S), although these proteins differ significantly in the electron affinity of their heme groups as measured by their anaerobic redox potentials. Dolphin Hb, a cooperative Hb with a redox potential like that of Hb S, produces less SNO-Hb, indicating that steric considerations outweigh effects of altered electron affinity at the active-site heme groups in control of SNO-Hb formation. Examination of oxygen binding at 5-20 mM heme concentrations revealed increases due to S-nitrosation in the apparent oxygen affinity of both Hb A(0) and Hb S, similar to increases seen at lower heme concentrations. As observed at lower heme levels, deoxygenation is not sufficient to trigger release of NO from SNO-Hb. A sharp increase in apparent oxygen affinity occurs for unmodified Hb S at concentrations above 12.5 mM, its minimum gelling concentration. This affinity increase still occurs in 30 and 60% S-nitrosated samples, but at higher heme concentration. This oxygen binding behavior is accompanied by decreased gel formation of the deoxygenated protein. S-nitrosation is thus shown to have an effect similar to that reported for other SH-group modifications of Hb S, in which R-state stabilization opposes Hb S aggregation.

Anemia, Sickle Cell↗
PubMed1999

Induction of negative regulators of haematopoiesis in human bone marrow cells by HLA-DR cross-linking.

Tumour necrosis factor-alpha (TNF alpha) is up-regulated by cross-linking of major histocompatibility complex (MHC) class II [human leucocyte antigen (HLA)-DR] antigens on monocytes. This is done by a bacterial superantigen or anti-HLA-DR monoclonal antibody (MAb). We have previously shown that HLA-DR cross-linking results in inhibition of haematopoiesis and apoptosis. TNF alpha acts as a negative regulator of haematopoiesis. Here we investigated whether HLA-DR-mediated inhibition of haematopoiesis involved TNF alpha and TNF alpha-dependent secondary signals. Anti-HLA-DR MAb H81.9 up-regulated TNF alpha, as well as transforming growth factor beta, interleukin (IL)-1beta and IL-6 in human marrow cells at the ribonucleic acid (RNA) and protein level. The effect on TNF alpha was investigated further. Up-regulation was blocked by herbimycin A, consistent with a tyrosine kinase-dependent mechanism. Up-regulation was also blunted by the soluble TNF-receptor fusion protein TNFR:Fc, suggesting an autocrine amplification loop. Following TNF alpha up-regulation, there was increased expression of Fas (CD95) and Fas-ligand (Fas-L). Up-regulation of Fas and Fas-L was blocked by TNFR:Fc. Furthermore, MAb H81.9-induced apoptosis was prevented by anti-TNF alpha MAb and by the soluble Fas receptor, Fas-Ig, providing further evidence that the TNF effect was mediated via Fas. At the transcriptional level, cross-linking of HLA-DR by MAb H81.9 affects nuclear localization of NFkappaB, which is involved in the transcription of TNF alpha. NFkappaB activity is modified by changes in cellular redox potential, and we have shown that H81.9 affects redox potential as determined by the generation of nitric oxide. These data show that HLA-DR-initiated signals are able to trigger a cascade of negative regulators of haematopoiesis. This model provides an opportunity to dissect signalling pathways that may be involved in the development of spontaneous marrow failure, and to devise interventions aimed at protecting haematopoiesis.

Antibodies, Monoclonal↗
PubMed2001

Binding studies between tetrathiafulvalene derivatives and cyclobis(paraquat-p-phenylene).

The complexation between a number of different pi-electron donating TTF derivatives and the pi-electron accepting tetracationic cyclophane cyclobis(paraquat-p-phenylene) (CBPQT(4+)) has been studied by (1)H NMR and UV-vis spectroscopy. The results demonstrate that the strength of association between the donors (TTF derivatives) and acceptor (CBPQT(4+)) is strongly dependent on the pi-electron donating properties (measured by the first redox potential ) of the TTF derivatives. However, the first redox potential () is not the only factor of importance. The extended pi-surface of the TTF derivatives also exerts a stabilizing influence upon complexation. The kinetics for the complexation-decomplexation were studied using (1)H NMR spectroscopy and are related to the bulkiness of the TTF derivatives. These effects may serve to improve the design of interlocked molecular systems, especially (bistable) molecular switches, in which CBPQT(4+) and a derivatized TTF unit are incorporated.

Journal Article↗
PubMed2001

Structures and comparison of the Y98H (2.0 A) and Y98W (1.5 A) mutants of flavodoxin (Desulfovibrio vulgaris).

The structures for two mutants at the Tyr98 site of Desulfovibrio vulgaris flavodoxin have been determined. The first, a tyrosine-to-histidine (Y98H) variant, was determined at the moderately high resolution of 2.0 A, while the tyrosine-to-tryptophan variant (Y98W) yielded very high resolution data (beyond 1.5 A) allowing a detailed look at the water structure, alternate side-chain conformations and the planarity of the FMN. Both structures were solved by molecular replacement beginning with the native (P2A) coordinates as a starting point. The Y98H variant of D. vulgaris flavodoxin crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 41.96, b = 61.45, c = 57.04 A, while the Y98W mutant adopts space group P2(1), with a = 41.29, b = 55.82, c = 32.52 A, beta = 100.68 degrees. Refinement for both mutants utilized PROLSQ followed by, for the high-resolution Y98W structure, anisotropic refinement as implemented in SHELXL. Final R factors of 17% for the Y98H mutant and 9.8% for the Y98W mutant were obtained. For the high-resolution (1.5 A) Y98W mutant, 31,010 unique reflections were collected from a single crystal. The final model includes 273 solvent molecules, with eight side chains assuming multiple conformations. At this resolution, the detailed conformation of the FMN can be observed, with both a bow and twist being noted. A comparison is made between the two mutants and the different oxidation states of the native flavodoxin. Although both mutants show similar E(2) (oxidized/semiquinone) one-electron redox potentials to the native, the E(1) (semiquinone/hydroquinone) redox potential for the Y98H mutant is significantly different from that of the Y98W variant and the native protein. The surprising similarity in the folding of the polypeptide chain 60--64 between the two mutants and the reduced states of the native is discussed. The interaction between O61 and N5 in the flavin is discussed because of the new conformation of this loop.

Amino Acid Substitution↗
PubMed1984

Antimicrobic effects of Lactobacillus fermentation on edible waste material contaminated with infected carcasses.

Survival of Newcastle disease virus (NDV) and Salmonella enteritidis serovar typhimurium (introduced as infected carcasses) in Lactobacillus-fermented edible waste material was studied to determine the ability of the fermentation to activate disease agents in carcasses. Two trials were conducted. In the 1st trial, the fermented wastes contained 20% infected carcasses (protein) consisting of equal numbers of chicken (NDV) and rat (Salmonella) carcasses, and the 2nd trial contained 40% carcasses (protein). Mixtures were incubated at 20 C, 30 C, and 40 C for 216 hours. Samples were obtained daily for quantitative virus and bacterial isolation. Temperature, pH, and redox potential were monitored. In both trials, pH and redox potential changes occurred between 24 and 48 hours depending on the incubation temperature. In both trials, NDV survived 4 days at 20 C, 2 days at 30 C, and 1 day at 40 C. Salmonella enteritidis serovar typhimurium survived 5 days at 20 C, 1 day at 30 C, and was not isolated from the 40 C samples after 24 hours in both trials.

Animal Feed↗
PubMed1987

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Alcaligenes↗
PubMed1991

Diurnal fluctuations of arterial ketone body ratio in normal subjects and patients with liver dysfunction.

To explore the metabolic aspects of chronic liver disease, diurnal changes of arterial ketone body ratio (acetoacetate/3-hydroxybutyrate), reflecting hepatic mitochondrial redox potential, were investigated in normal subjects, patients with chronic liver disease (Child's class A or B), and patients with hepatic failure (Child's class C). Ketone body ratio in normal subjects increased after breakfast from 0.96 to 2.00, after lunch from 2.17 to 2.38, and after dinner from 1.23 to 2.55 with blood glucose level ranging from 103 to 141 mg/dL (5.7 to 7.8 mmol/L). By contrast, the ketone body ratio in the Child A or B group changed little and remained within a range of 0.70-1.35 despite a large change in blood glucose level from 102 to 176 mg/dL (5.7 to 9.8 mmol/L). Ketone body ratio in Child's class C remained near or below 0.4 with no response to glucose administration, despite a marked elevation in blood glucose level. These results indicate that hepatic mitochondrial redox potential undergoes diurnal changes in sharp response to meals in normal liver function but that these fluctuations are absent in patients with liver damage (Child's class A, B, and C). Furthermore, it remains at low levels in severe liver failure (Child's class C). It is also suggested that hepatic mitochondrial redox potential plays an important role in the regulation of blood glucose levels.

Acetoacetates↗
PubMed1986

Oxidation-reduction properties of glycolate oxidase.

This is the first report of the redox potentials of glycolate oxidase. The pH dependence of the redox behavior as well as the effects of activators and inhibitors was studied. At pH 7.1 in 10 mM imidazole-chloride, Eo1' (EF1ox/EF1-.) is -0.033 +/- 0.010 V and Eo2' (EF1-./EF1redH-) is -0.017 +/- 0.017 V vs. the standard hydrogen electrode at 10 degrees C. A maximum of 29% flavin mononucleotide (FMN) anion radical is stabilized at half-reduction at pH 7.1 and 10 degrees C. Both redox couples of glycolate oxidase are pH-dependent from pH 7 to pH 9, and the FMN anion radical is stabilized in this range. The redox potentials of glycolate oxidase are shifted markedly positive of those of unbound FMN, consistent with the enzyme's function. The midpoint potential of glycolate oxidase is more positive than that of the glyoxalate/glycolate couple, and two-electron reduction of glycolate oxidase is thermodynamically favorable. The redox behavior of glycolate oxidase markedly contrasts that of other flavoprotein oxidases. For most flavoprotein oxidases, Eo1' is independent of pH from pH 7 to pH 9 and is much more positive than Eo2', which is pH-dependent. We present a mechanism that suggests a structural basis for the positive shifts and pH dependence of both Eo1' and Eo2' of glycolate oxidase.

Alcohol Oxidoreductases↗
PubMed1983

Characterization of succinate dehydrogenase from Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy.

Low-temperature electron spin resonance spectroscopy has been used to study the biophysical properties of succinate dehydrogenase from the gram-positive bacterium Micrococcus luteus. The paramagnetic redox centres of the enzyme were identified in a succinate-dehydrogenase--antigen complex, which had been purified with the aid of monospecific serum from membranes solubilized with Triton X-100. The centres were characterized in further detail using the membrane-bound and Triton-solubilized forms of the enzyme. These studies distinguished two types of iron-sulphur centres, viz. a [4Fe-4S]3+ cluster displaying a narrow signal at g = 2.01 in the oxidized state (conventionally termed centre S-3) and a [2Fe-2S )0 cluster with an axial signal at g = 2.03 and 1.93 in the reduced state (conventionally termed centre S-1). Centre S-3 had a mid-point redox potential of +10 mV, a comparatively low value for this type of cluster. The behaviour of the g = 1.93 signal of centre S-1 was a complex function of the redox potential, microwave power and temperature of measurement. When measured at low power (i.e. non-saturating conditions), the intensities observed for the g = 1.93 signal poised at various critical potentials in the redox titration were similar. However, the corresponding intensities differed markedly at high power, where conditions were saturating. It is proposed that under saturating conditions the spin-lattice relaxation of the [2Fe-2S] cluster S-1 (mid-point potential +70 mV) is enhanced by centre S-3 between the potential range +10-+70 mV and by an ESR-silent centre, termed centre S-2, with a mid-point potential of -295 mV.

Chemical Precipitation↗
PubMed1998

Construction and analysis of a semi-quantitative energy profile for the reaction catalyzed by the radical enzyme galactose oxidase.

Galactose oxidase (GOase) is a mononuclear type 2 copper enzyme which oxidizes primary alcohols to aldehydes using molecular oxygen (RCH2OH + O2 = RCHO + H2O2). An unusual crosslink between tyrosine 272 and cysteine 228 provides a modified tyrosine radical site which acts as a ligand for the active site copper and is believed to act as a one-electron redox center. The single active site copper is believed to act as a second one-electron redox center. The use of the tyrosine one-electron redox center and the copper one-electron redox center allows removal of two electrons from alcohol substrate for subsequent transfer to molecular oxygen. Previously, we and others have proposed a detailed step-by-step radical mechanism for the reaction catalyzed by galactose oxidase. The catalytic cycle can be divided into two half reactions. The first half reaction entails transfer of two electrons and two protons from the alcohol substrate to the enzyme to form aldehyde product and two-electron-reduced enzyme (one electron at the tyrosine center and one at the copper center). The second half reaction entails transfer of two electrons and two protons from the two-electron-reduced enzyme to O2 to form H2O2 product and regenerate fully oxidized catalytically active enzyme ready for another catalytic cycle. In this paper, we describe the construction of a semi-quantitative energy profile for this radical mechanism. Several significant points emerge from this analysis. One point is the prediction that galactose oxidase should have an unusually low redox potential for copper, to our knowledge lower than any other redox active copper protein. Another point is that the distorted or entatic copper site causes the unusually low redox potential. A final point is that crosslinking of tyrosine 272 and cysteine 228 alters the redox properties of the tyrosine center to enhance catalysis compared to what would be expected for a normal tyrosine.

Energy Transfer↗
PubMed1985

Redox study of electron donation to P-680 in Photosystem II.

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.

Chlorophyll↗