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PubMed1992

The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1996

Effects of pteridines on chloramine-T-induced growth inhibition in E. coli strains: correlations with molecular structure.

Little is known about the biological significance of most pteridines, despite their ubiquitous occurrence in living cells. Seventeen different pteridines were tested for their ability to modulate the growth inhibitory effect of the disinfectant chloramine-T on three different strains of Escherichia coli bacteria. We found striking differences between the pteridine derivatives: whereas aromatic pterins with a hydroxy function at side chain atom C2' increased the growth inhibition, those with a 7,8-dihydro structure exerted a suppressive effect. These results are in excellent agreement with previously observed effects of pteridine derivatives on chloramine-T-induced luminol-dependent chemiluminescence, and together, are highly suggestive of a general interaction of these compounds with oxygen or chlorine free radicals. This interaction is likely to have biological significance and might offer an explanation for the widespread occurrence of pteridines.

Anti-Infective Agents, Local↗

PubMed1996

Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects.

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.

Biopterins↗

PubMed2005

A nhaD Na+/H+ antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes.

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.

Amino Acid Sequence↗

PubMed2006

Cell biology of molybdenum.

The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing diverse key reactions in the global carbon, sulfur and nitrogen metabolism. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In eukaryotes, the most prominent Mo-enzymes are (1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite, (2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production, (3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms also (4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation. All Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the post-translational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from l-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases.

Aldehyde Oxidase↗

PubMed2004

Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo.

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.

Acetylcholinesterase↗

PubMed2004

Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha.

Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.

Amino Acid Sequence↗

PubMed2004

Interference of 7,8-dihydroneopterin with peroxynitrite-mediated reactions.

By in vitro studies 7,8-dihydroneopterin, which is secreted by macrophages stimulated by interferon-gamma, was reported to be a radical scavenger as well as a prooxidative agent depending on the experimental settings. In this study, we investigated the interference of 7,8-dihydroneopterin with peroxynitrite mediated reactions by different analytical procedures. Luminol chemiluminescence and oxidation of the spin probe 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine induced by peroxynitrite were inhibited by 7,8-dihydroneopterin. On the other hand, we found that 7,8-dihydroneopterin very efficiently inhibits nitration of tyrosine by peroxynitrite. Hydroxylation, however, was rather enhanced than inhibited, suggesting that 7,8-dihydroneopterin reacts in quite different manner with the intermediates generated from peroxynitrite. We provide the first evidence that a pterin radical is formed from a dihydropterin using EPR spectroscopy and 2,2,4-trimethyl-2H-imidazole-1-oxide as a spin trap. We conclude that 7,8-dihydroneopterin while being a weak scavenger of superoxide acts as a very efficient inhibitor of tyrosine nitration induced by peroxynitrite.

Electron Spin Resonance Spectroscopy↗

PubMed2004

Regulation of tyrosinase by tetrahydropteridines and H2O2.

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.

Binding Sites↗

PubMed2006

The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus.

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.

Amino Acid Sequence↗

PubMed2005

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.

Amino Acid Sequence↗

PubMed2005

Crystal structure of Trypanosoma cruzi pteridine reductase 2 in complex with a substrate and an inhibitor.

Reduced pteridines are required for a number of important cellular functions. Trypanosomatid parasites, unlike their mammalian hosts, are pteridine auxotrophs and salvage the precursor pteridines from the host and reduce them to the respective biologically active tetrahydro forms using parasite-encoded enzymes. These enzymes may offer selective drug targets. In Leishmania, pteridine reductase 1 (PTR1), the primary enzyme for reducing pterins, is also responsible for resistance to antifolate drugs. Typically, PTR1 is more active with fully oxidized biopterin and folate than with their reduced counterparts. We have identified an enzyme, TcPTR2 of Trypanosoma cruzi, which though very similar to PTR1 in its primary sequence, can reduce only dihydrobiopterin and dihydrofolate and not oxidized pteridines. The structures of an inhibitor (methotrexate) and a substrate (dihydrofolate) complex of this enzyme demonstrate that the orientation of the substrate and the inhibitor in the active site of TcPTR2 are different from each other. However, the orientation of each ligand is similar to that of the corresponding ligand in Leishmania major PTR1 complexes.

Amino Acid Sequence↗

PubMed2006

Sepiapterin reductase deficiency: clinical presentation and evaluation of long-term therapy.

Sepiapterin reductase deficiency has recently been recognized as a treatable, inborn error of pterin metabolism. This investigation is the first long-term clinical study demonstrating impressive positive, long-term effects of treatment in two cases of sepiapterin reductase deficiency after 2 and 5 years of treatment respectively. The two patients were not diagnosed before 7 and 13 years of age. These results highlight the importance of cerebrospinal fluid neurotransmitter investigations in childhood encephalopathy, in cases of unexplained early-onset neurologic handicap. Such a widened approach to the diagnostic efforts in early-onset encephalopathy with motor delay during childhood is important, as we have at our disposal a simple and effective treatment.

5-Hydroxytryptophan↗

PubMed2004

Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani.

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.

Amino Acid Sequence↗

PubMed2005

Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase.

The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)-dihydropterin pyrophosphokinase (PPPK)-dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.

Amino Acid Sequence↗

PubMed2005

Incidence of BH4-responsiveness in phenylalanine-hydroxylase-deficient Italian patients.

BACKGROUND: Hyperphenylalaninemia (HPA) is an inherited metabolic disorder due to deficiency of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4). BH4-responsiveness in PAH-deficient HPA is a recently described characteristic of most milder phenotypes. BH4-responsive patients show reduction of plasma phenylalanine (phe) levels after oral administration of BH4. AIM: Determination of the incidence of BH4-responsiveness among a non-selected, cohort population of PAH-deficient hyperphenylalaninemic patients and evaluation of phenotype-genotype correlations. PATIENTS AND METHODS: All patients born in Lombardy (Italy) between January 2000 and December 2004, and affected by HPA (107 patients) were classified after BH4 loading test, analysis of urinary pterins, and determination of DHPR activity in blood, and investigated for BH4-responsiveness. 6R-BH4 (20 mg/kg) was administered orally as a single dose and plasma samples were obtained at time-points 0, 4, 8, and 24 h after BH4 administration. In patients with basal plasma phe levels <or=360 mmol/L a combined phe (100 mg phe/kg) and BH4 (20 mg/kg) loading test was performed. Patients were defined "responsive to BH4" when plasma phe levels decreased by 30% 8h after oral BH4 administration. RESULTS: BH4 significantly lowered blood phe levels in 91 (85%) of 107 patients affected by PAH-deficient HPA. Most responsive patients were affected by mild HPA (77%), a smaller percentage by mild (7%) and moderate (7%) phenylketonuria (PKU). One patient with classical PKU was responsive to BH4. Eighteen mutations were found to be associated to the BH4-responsive phenotype. CONCLUSIONS: BH4-responsiveness is shown by a consistent number of PAH-deficient hyperphenylalaninemic patients and seems to be common in milder phenotypes. Genotype is not the only factor determining BH4-responsiveness.

Biopterins↗

PubMed2006

Can the DCoHalpha isozyme compensate in patients with 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH deficiency?

4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1alpha (HNF1). Patients with a deficiency of dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, dehydratase/DCoHalpha, has been detected which shares 60% identity with dehydratase/DCoH. This investigation was undertaken to ascertain if dehydratase/DCoHalpha has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHalpha demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHalpha, demonstrates that DCoHalpha does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The dehydratase activity of each isoform was measured by a direct spectrophotometric assay. Km and Vmax for DCoHalpha were both 2-3 times higher than for DCoH, thus leaving the catalytic efficiency (Vmax/Km) the same for both enzymes. In conclusion, the properties of dehydratase/DCoHalpha are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in dehydratase/DCoH.

DNA↗

PubMed2002

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.

Amino Acid Sequence↗