Reductive Titrations Research Articles

His92 and His110 selectively affect different heme centers of adrenal cytochrome b 561

Adrenal cytochrome b 561 (cyt b 561 ), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b 561 (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31–40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b H) peak were seen with mutation of His92; the largest changes in the low-potential (b L) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (b H) but not the g = 3.7 signal (b L). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b H transition; mutations in His110 produced the largest decreases in Δ A 561 for the b L transition. These results indicate that His92 can be considered part of the b H heme center, and His110 part of the b L heme center, in adrenal cyt b 561 .

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Vitamin B(12), the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6A resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Thermodynamic Redox Properties Governing the Half-Reduction Characteristics of Histamine Dehydrogenase fromNocardioides simplex

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers' redox potential.

Cadmium telluride quantum dots as pH-sensitive probes for tiopronin determination

The pH-sensitive cadmium telluride (CdTe) quantum dots (QDs) were used as proton probes for tiopronin determination. Based on the fluorescence quenching of CdTe QDs caused by tiopronin, a simple, rapid and specific quantitative method was proposed. Under the optimal conditions, the calibration plot of ln( F 0/ F) with concentration of tiopronin was linear in the range of 0.15–20 μg mL −1(0.92–122.5 μmol L −1) with correlation coefficient of 0.998. The limit of detection (LOD) (3 σ/ k) was 0.15 μg mL −1(0.92 μmol mL −1). The content of tiopronin in pharmaceutical tablet was determined by the proposed method and the result agreed with that obtained from the oxidation–reduction titration method and the claimed value.

A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

Thermodynamic Basis for Redox Regulation of the Yap1 Signal Transduction Pathway

The Yap1 oxidative stress signal transduction pathway found in Saccharomyces cerevisiae is redox-regulated. We have examined the thermodynamic basis of the disulfide/dithiol couples that are involved in the regulation of this pathway. The oxidized form of the Yap1 redox domain (Yap1-RD) fragment, derived from the Yap1 transcription factor, contains two disulfide bonds, one between Cys303 and Cys598 and one between Cys310 and Cys629. Oxidation-reduction titrations reveal the presence of two separate two-electron redox couples in Yap1-RD, with redox midpoint potentials (E(m)) of -155 and -330 mV, respectively, at pH 7.0. We measured E(m) values of -275 and -265 mV for the two cytoplasmic S. cerevisiae thioredoxins, Trx1 and Trx2, respectively, both at pH 7.0. Last, we measured an E(m) value of -255 mV for the Cys36-Cys82 disulfide bond at pH 6.0 in the glutathione peroxidase-like enzyme, oxidant receptor protein (Orp1). We were unable to obtain satisfactory redox titration data for Orp1 at pH 7.0, but if the redox-active disulfide of Orp1 exhibits the -59 mV per pH unit dependence for E(m) typical of protein disulfides in this pH region, an E(m) value of -315 mV can be estimated for Orp1 at pH 7.0 by extrapolation. Together, these data suggest that, at physiological ratios of Trx(ox)/Trx(red), the reduction of both the E(m) = -315 mV disulfide of Orp1 and the E(m) = -330 mV disulfide of Yap1 by either Trx1 or Trx2 would be thermodynamically possible.

Determination of the Bulk Cobalt Valence State of Co-Perovskites Containing Surface-Adsorbed Impurities

We used thermogravimetric hydrogen reduction and iodometric titration to determine the bulk valence state of cobalt in Co-perovskites containing surface carbonate hydroxide or hydroxyl groups. It could be shown that thermogravimetric hydrogen reduction experiments are very sensitive to volatile surface groups, but due to their volatility, they can be specified and the bulk valence state of cobalt can still be deduced from these experiments. The iodometric titration is less sensitive to small volatile surface impurities, but precaution has to be taken that oxygen or iodine does not escape from the solution during dissolution of the sample. Best results were obtained if the sample was titrated during dissolution in a closed argon floated titration apparatus. We tested the two methods using LaCoO3 perovskite as a sample with a known valence state. Both methods delivered satisfactory results, and the valence state could be determined with an accuracy of better than 1%.

The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H(+)/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Evaluation of certified reference materials for oxidation–reduction titration by precise coulometric titration and volumetric analysis

The accuracy and uncertainty of coulometric titration of Japanese certified reference materials (CRMs) for oxidation–reduction titration were examined in this study. The results for potassium dichromate, sodium oxalate, and potassium iodate are presented. Potassium dichromate was directly determined by coulometric titration, and sodium oxalate and potassium iodate were determined by volumetric analysis using potassium dichromate assigned by coulometry. The uncertainty of the method was investigated by examining the dependency on the sample size and on the electrolysis current. Changes in the titration parameters did not result in any significant effects on the titration results. The coulometric system used primarily consists of a constant current source, timer, switching circuit, indicator unit, and voltmeter. They were controlled using the coulometry software by a PC/AT compatible computer. A highly automated coulometric system achieves repeatabilities of less than one part in 30,000 ( k = 2) and uncertainties of less than one part in 15,000 ( k = 2). In addition, using volumetric method, SI units traceable sodium oxalate and potassium iodate (purity standards for redox reaction) CRMs were developed. Reference materials for volumetric analysis are the most basic substances used in analytical chemistry. These materials are analyzed by several analytical methods and are produced globally; however, their purities have not been compared at the international level. Therefore, the relationship between the purity and reliability of these materials has not yet been established. In this paper, we determine the relationship between these parameters by titrating each material obtained from different laboratories.

Cost-effectiveness analysis of darbepoetin after switch from epoetin-beta in patients on hemodialysis: an Italian single-center retrospective study

Darbepoetin (DARB), a human erythropoietin with improved pharmacological parameters (longer half life and a dose-response relationship independent from the route of administration) is a new treatment option for chronic nephropathy-induced anemia that in many Italian dialysis centers is substituting older erythropoietins, mainly due to greater ease of use. At current prices and at the theoretical and recommended 200:1 dose conversion factor, DARB pharmaceutical cost ranges from –9% to +17% of EPO cost. In this monocentric study, real drug consumption and related actual costs of a pre-switch semester with EPO were analysed and compared to the first two post-switch semesters with DARB therapy. Furthermore, erythropoetic response was recorded and related to drug exposition levels, in order to establish a real life EPO:DARB dose conversion factor. In this experience, the conversion factor was estimated in at least 250-280:1, depending on the method adopted for the estimation. The initial dose, chosen on the basis of the recommended conversion factor, induced an excessive erythropoietic response, which was generally followed by a dose reduction and successive titration to target desired Hb levels, generating the spiraliform dose-response curve associated with delayed feed-back mechanisms. The pharmacoeconomical analysis, conducted in the perspective of the public health service, indicated that the choice of substituting DARB for EPO in the treatment of chronic kidney failure-associated anemia is univocally convenient, the former being less costly and more effective.

Nitric-oxide Synthase Output State

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

Switching Pyridine Nucleotide Specificity in P450 BM3: MECHANISTIC ANALYSIS OF THE W1046H AND W1046A ENZYMES

Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K(m)(NADPH)/k(cat)/K(m)(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant approximately 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).

Mechanistic Studies of Rhodobacter sphaeroides Me2SO Reductase

Studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides have yielded new insight into its catalytic mechanism. A series of reductive titrations, performed over the pH range 6-10, reveal that the absorption spectrum of reduced enzyme is highly sensitive to pH. The reaction of reduced enzyme with dimethyl sulfoxide is found to be clearly biphasic throughout the pH range 6-8 with a fast, initial substrate-binding phase and substrate-concentration independent catalytic phase. The intermediate formed at the completion of the fast phase has the characteristic absorption spectrum of the established dimethyl sulfoxide-bound species. Quantitative reductive and oxidative titrations of the enzyme demonstrate that the molybdenum center takes up only two reducing equivalents, implying that the two pyranopterin equivalents of the molybdenum center are not formally redox active. Finally, the visible spectrum associated with the catalytically relevant "high-g split" Mo(V) species has been determined. Spectral deconvolution and EPR quantitation of enzyme-monitored turnover experiments with trimethylamine N-oxide as substrate reveal that no substrate-bound intermediate accumulates and that Mo(V) content remains near unity for the duration of the reaction. Similar experiments with dimethyl sulfoxide show that significant quantities of both the Mo(V) species and the dimethyl sulfoxide-bound complex accumulate during the course of reaction. Accumulation of the substrate-bound complex in the steady-state with dimethyl sulfoxide arises from partial reversal of the physiological reaction in which the accumulating product, dimethyl sulfide, reacts with oxidized enzyme to yield the substrate-bound intermediate, a process that significantly slows turnover.

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH 2) through the cytochrome b 6 f complex (Cyt b 6 f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light–dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700 + and PC + was recorded as leaf transmission measured differentially at 810–950 nm. The signal was deconvoluted into PC + and P700 + components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1–15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225–234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b 6 f to the PSI donors. A significant down-regulation of Cyt b 6 f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b 6 f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light–dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e − transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.

Interface Defect Chemistry and Effective Conductivity in Polycrystalline Cerium Oxide

Polycrystalline cerium oxide exhibits increasing electronic and decreasing ionic conductivity upon reduction of the grain size. In the present study, the origin of this effect was examined. Temperature-programmed reduction (TPR) and oxygen titration measurements on nanocrystalline cerium oxide revealed a large excess oxygen deficiency associated with the surface. Using a two-phase model for the combined system of the bulk phase in equilibrium with a surface layer, this enhanced oxygen deficiency could be explained by a reduced binding energy of surface oxygen ions in agreement with results from atomistic computer simulations. The model also revealed that this segregation of oxygen vacancies is the origin of an intrinsic space charge potential. Translating this effect to polycrystalline cerium oxide and taking into account the segregation of dopants and the accumulation/depletion of charge carriers, it was possible to model the grain size dependence of electrical conductivity and thermopower of polycrystalline cerium oxide. A straightforward 1-dimensional numerical model and a change from Boltzmann to Fermi-Dirac statistics allowed to calculate the conductivity of heavily doped polycrystalline cerium oxide for grain sizes in the range of 5–10,000 nm and acceptor concentrations up to 20%. Using this approximation, the effect of grain size on mixed ionic/electronic conductivity and the electrolytic domain boundary was investigated.

Preparation and characterization of a (Cu, Zn)-pMMO from Methylococcus capsulatus (Bath)

We report the preparation of a (Cu, Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu, Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu, Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu, Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu, Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.

The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter capsulatus Xanthine Dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.

Reductive titration of photosystem I and differential extinction coefficient of P700 + at 810–950 nm in leaves

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e −) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e − carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1–15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch ( Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e − generated in PSII by the flash was measured as four times O 2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700 + and PC + components using the abovementioned oxidative titration method. The P700 + component was related to the absolute number of e − that reduced the P700 + to calculate the extinction coefficient. The effective differential extinction coefficient of P700 + at 810–950 nm was 0.40±0.06 (S.D.)% of transmittance change per μmol P700 + m −2 or 17.6±2.4 mM −1 cm −1. The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (±14% S.D.) is mainly caused by light-scattering properties of the leaf.