EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe–4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to −500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe–4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Transfer of exogenous cholesterol to microsomes of hepatocytes investigated with [3H]desmosterol tracer.

The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.     

Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion

In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the “high g” species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) “high g” resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe–4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin–spin interactions between the Mo(V) ion and the [4Fe–4S]^1 + cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe–4S]^1 + centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe–4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.     

Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.     

Molybdenum Availability Is Key to Nitrate Removal in Contaminated Groundwater Environments

The concentrations of molybdenum (Mo) and 25 other metals were measured in groundwater samples from 80 wells on the Oak Ridge Reservation (ORR) (Oak Ridge, TN), many of which are contaminated with nitrate, as well as uranium and various other metals. The concentrations of nitrate and uranium were in the ranges of 0.1 μM to 230 mM and <0.2 nM to 580 μM, respectively. Almost all metals examined had significantly greater median concentrations in a subset of wells that were highly contaminated with uranium (≥126 nM). They included cadmium, manganese, and cobalt, which were 1,300- to 2,700-fold higher. A notable exception, however, was Mo, which had a lower median concentration in the uranium-contaminated wells. This is significant, because Mo is essential in the dissimilatory nitrate reduction branch of the global nitrogen cycle. It is required at the catalytic site of nitrate reductase, the enzyme that reduces nitrate to nitrite. Moreover, more than 85% of the groundwater samples contained less than 10 nM Mo, whereas concentrations of 10 to 100 nM Mo were required for efficient growth by nitrate reduction for two Pseudomonas strains isolated from ORR wells and by a model denitrifier, Pseudomonas stutzeri RCH2. Higher concentrations of Mo tended to inhibit the growth of these strains due to the accumulation of toxic concentrations of nitrite, and this effect was exacerbated at high nitrate concentrations. The relevance of these results to a Mo-based nitrate removal strategy and the potential community-driving role that Mo plays in contaminated environments are discussed.     

Nitrate reductase from Chlorella vulgaris. Reaction with manganese (III) pyrophosphate and with ferric o-phenanthroline.

Millimolar concentrations of tervalent manganese pyrophosphate can partially activate nitrate reductase which has been inactivated with NADH and HCN. The tervalent manganese complex is nevertheless not reduced by NADH in the presence of the enzyme, that is, it is not a substrate for the diaphorase moiety of the nitrate reductase. Ferric o-phenanthroline, on the other hand, is a good diaphorase substrate, but fails to activate the inactive enzyme.     

chlD gene function in molybdate activation of nitrate reductase.

chlD mutants of Escherichia coli lack active nitrate reductase but form normal levels of this enzyme when the medium is supplemented with 10-3 M molybdate. When chlD mutants were grown in unsupplemented medium and then incubated with molybdate in the presence of chloramphenicol, they formed about 5% the normal level of nitrate reductase. Some chlD mutants or the wild type grown in medium supplemented with tungstate accumulated an inactive protein which was electrophoretically identical to active nitrate reductase. Addition of molybdate to those cells in the presence of chloramphenicol resulted in the formation of fully induced levels of nitrate reductase. Two chlD mutants, including a deletion mutant, failed to accumulate the inactive protein and to form active enzyme under the same conditions. Insertion of 99-Mo into the enzyme protein paralleled activation; 185-W could not be demonstrated to be associated with the accumulated inactive protein. The rates of activation of nitrate reductase at varying molybdate concentrations indicated that the chlD gene product facilitates the activation of nitrate reductase at concentrations of molybdate found in normal growth media. At high concentrations, molybdate circumvented this function in chlD mutants and appeared to activate nitrate reductase by a mass action process. We conclude that the chlD gene plays two distinguishable roles in the formation of nitrate reductase in E. coli. It is involved in the accumulation of fully induced levels of the nitrate reductase protein in the cell membrane and it facilitates the insertion of molybdenum to form the active enzyme.     

Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster

Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.     

Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Binding of C5 Protein to P RNA Enhances the Rate Constant for Catalysis for P RNA Processing of Pre-tRNAs Lacking a Consensus G(+ 1)/C(+ 72) Pair

The RNA subunit of the ribonucleoprotein enzyme ribonuclease P (RNase P (P RNA) contains the active site, but binding of Escherichia coli RNase P protein (C5) to P RNA increases the rate constant for catalysis for certain pre-tRNA substrates up to 1000-fold. Structure-swapping experiments between a substrate that is cleaved slowly by P RNA alone (pre-tRNA^f-met605) and one that is cleaved quickly (pre-tRNA^met608) pinpoint the characteristic C(+ 1)/A(+ 72) base pair of initiator tRNA^f-met as the sole determinant of slow RNA-alone catalysis. Unlike other substrate modifications that slow RNA-alone catalysis, the presence of a C(+ 1)/A(+ 72) base pair reduces the rate constant for processing at both correct and miscleavage sites, indicating an indirect but nonetheless important role in catalysis. Analysis of the Mg²⁺ dependence of apparent catalytic rate constants for pre-tRNA^met608 and a pre-tRNA^met608 (+ 1)C/(+ 72)A mutant provides evidence that C5 promotes rate enhancement primarily by compensating for the decrease in the affinity of metal ions important for catalysis engendered by the presence of the CA pair. Together, these results support and extend current models for RNase P substrate recognition in which contacts involving the conserved (+ 1)G/C(+ 72) pair of tRNA stabilize functional metal ion binding. Additionally, these observations suggest that C5 protein has evolved to compensate for tRNA variation at positions important for binding to P RNA, allowing for tRNA specialization.     

Multifrequency cw-EPR investigation of the catalytic molybdenum cofactor of polysulfide reductase from<Emphasis Type="Italic"> Wolinella succinogenes</Emphasis>

Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different Mo(V) states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the Mo(V) states, based on the experimentally observed shift of the g(av) value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the Mo(V) G-tensor supports this assignment.     

Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site

The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli.     

Direct catalytic electrochemistry of sulfite dehydrogenase: Mechanistic insights and contrasts with related Mo enzymes

Under hydrodynamic electrochemical conditions with slow cyclic voltammetry sweep rates we have been able to probe catalytic events at the molybdenum active site of sulfite dehydrogenase (SDH) from Starkeya novella adsorbed on an edge plane graphite electrode within a polylysine film. The electrochemically driven catalytic behaviour of SDH mirrors that seen in solution assays suggesting that the adsorbed enzyme retains its native activity. However, at high sulfite concentrations, the voltammetric waveform transforms from the expected sigmoidal profile to a peak-shaped response, similar to that reported for the molybdenum enzymes DMSO reductase and nitrate reductase (NarGHI and NapAB) where a redox reaction at the active site has been associated with a switch to lower activity at high overpotentials. This is the first time a similar phenomenon has been observed in a Mo-containing oxidase/dehydrogenase, which raises a number of interesting mechanistic problems. The potential at which the activity of SDH becomes attenuated only emerges at saturating substrate conditions and occurs at a potential (ca. + 320mV vs NHE) well removed from any known redox couple in the enzyme. These results cannot be explained by the same mechanism adopted for DMSO reductase and nitrate reductase catalysis.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Architecture of NarGH reveals a structural classification of Mo-bisMGD enzymes

The structure of the catalytic and electron-transfer subunits (NarGH) of the integral membrane protein, respiratory nitrate reductase (Nar) has been determined to 2.0 A resolution revealing the molecular architecture of this Mo-bisMGD (molybdopterin-guanine-dinucleotide) containing enzyme which includes a previously undetected FeS cluster. Nar, together with the related enzyme formate dehydrogenase (Fdh-N), is a key enzyme in the generation of proton motive force across the membrane in Escherichia coli nitrate respiration. A comparative study revealed that Nar and Fdh-N employ different approaches for acquiring substrate, reflecting different catalytic mechanisms. Nar uses a very narrow and nonpolar substrate-conducting cavity with a nonspecific substrate binding site, whereas Fdh-N accommodates a wider, positively charged substrate-conducting cavity with a more specific substrate binding site. The Nar structure also demonstrates the first example of an Asp side chain acting as a Mo ligand providing a structural basis for the classification of Mo-bisMGD enzymes.     

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Redox potential of chlorophyll d in vitro

Chlorophyll (Chl) d is a major chlorophyll in a novel oxygenic prokaryote Acaryochloris marina. Here we first report the redox potential of Chl d in vitro. The oxidation potential of Chl d was + 0.88 V vs. SHE in acetonitrile; the value was higher than that of Chl a (+ 0.81 V) and lower than that of Chl b (+ 0.94 V). The oxidation potential order, Chl b > Chl d > Chl a, can be explained by inductive effect of substituent groups on the conjugated π-electron system on the macrocycle. Corresponding pheophytins showed the same order; Phe b (+ 1.25 V) > Phe d (+ 1.21 V) > Phe a (+ 1.14 V), but the values were significantly higher than those of Chls, which are rationalized in terms of an electron density decrease in the π-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was found to be the lowest among Chls and Phes. The results will help us to broaden our views on photosystems in A. marina.     

Reversible Inactivation of Nitrate Reductase by NADH and the Occurrence of Partially Inactive Enzyme in the Wheat Leaf

Nitrate reductase from wheat (Triticum aestivum L. cv Bindawarra) leaves is inactivated by pretreatment with NADH, in the absence of nitrate, a 50% loss of activity occurring in 30 minutes at 25°C with 10 micromolar NADH. Nitrate (50 micromolar) prevented inactivation by 10 micromolar NADH while cyanide (1 micromolar) markedly enhanced the degree of inactivation.

A rapid reactivation of NADH-inactivated nitrate reductase occurred after treatment with 0.3 millimolar ferricyanide or exposure to light (230 milliwatts per square centimeter) plus 20 micromolar flavin adenine dinucleotide. When excess NADH was removed, the enzyme was also reactivated by autoxidation. Nitrate did not influence the rate of reactivation.

Leaf nitrate reductase, from plants grown for 12 days on 1 millimolar nitrate, isolated in the late photoperiod or dark period, was activated by ferricyanide or light treatment. This suggests that, at these times of the day, the nitrate reductase in the leaves of the low nitrate plants is in a partially inactive state (NADH-inactivated). The nitrate reductase from moisture-stressed plants showed a greater degree of activation after light treatment, and inactive enzyme in them was detected earlier in the photoperiod.

     

Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH

Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKo(MoVI), pKo(MoV) less than 5.5; pKr(MoV), pKr(MoIV) greater than 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0.(ABSTRACT TRUNCATED AT 250 WORDS)