Radiation inactivation of assimilatory NADH:nitrate reductase from Chlorella. Catalytic and physical sizes of functional units

Assimilatory NADH:nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and Mo6+ per 100-kDa subunit. At low protein concentrations, this tetramer dissociates to a fully active dimer. To further elucidate the possible relationship between quaternary structure and activity, the functional size of nitrate reductase was determined by radiation inactivation analysis at high and low concentrations of enzyme where the principal physical species would be either tetrameric or dimeric, respectively. In both cases, the size obtained by this method was 100 kDa, suggesting that each subunit in the tetramer or dimer can function independently. These results confirm earlier results which indicated that the subunits are identical and that each contains a full complement of prosthetic groups. We also found that the functional sizes of the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase were fractions (approximately 58 kDa, 47 kDa, and 28 kDa, respectively) of the subunit molecular mass, suggesting that these domains are functionally independent.     

Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains.

Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.     

Escherichia coli nitrate reductase subunit A: its role as the catalytic site and evidence for its modification.

Subunits A and B were isolated from purified nitrate reductase by preparative electrophoresis in low levels of sodium dodecyl sulfate. Nonheme iron and low levels of molybdenum were associated with isolated subunit A but not with isolated subunit B. After dialysis against a source of molybdenum cofactor, subunit A regained tightly bound molybdenum and concomitantly regained enzyme activity and reactivity with anti-nitrate reductase antiserum. Subunit B neither bound cofactor nor regained activity or reactivity with antiserum. These data indicate that subunit A contains the active site of the enzyme. Subunit A was also found to be modified posttranslationally in a similar fashion as is subunit B. This was determined by comparison of partial proteolytic digests and amino acid analyses of A subunits from precursor and membrane-bound forms of nitrate reductase.     

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.     

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo^(IV) or Mo^(V) follow this path.     

Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.     

Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.     

Limited proteolysis of the nitrate reductase from spinach leaves

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.     

Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities

The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.     

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.     

Structure-based alteration of substrate specificity and catalytic activity of sulfite oxidase from sulfite oxidation to nitrate reduction

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 ? resolution, respectively.     

Sulfite oxidase from Merluccius productus

Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was purified 482-fold from liver of the Pacific hake Merluccius productus. The molecular weight of the enzyme was found to be 120 000 by gel exclusion chromatography on Sephadex G-100. Electrophoretic analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gel revealed that the enzyme was composed of two subunits whose molecular weight was estimated to be 60 000. The pH optimum of the enzyme was 8.7; Ks for sulfite, 2.5 x 10(-5) M; and that for cytochrome c, 3.6 x 10(-7) M. The enzyme elicited an EPR signal at g = 1.97 characteristic of pentavalent molybdenum. Colorimetric analysis also disclosed that the enzyme contained 2 mol each of heme and molybdenum per mol of protein. This fish liver homogenate in isotonic sucrose solution was fractionated by differential centrifugation into nuclei, mitochondria, microsomes and supernatant (100 000 X g). The major portion of sulfite oxidase activity was found in mitochondria. The sulfite oxidase activity was markedly high in liver and kidney, as compared with that in heart, spleen, muscle, gill and eye.     

Identification and biochemical characterization of Arabidopsis thaliana sulfite oxidase. A new player in plant sulfur metabolism

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC ). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K(m) values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants.     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

A simplifed functional version of the Escherichia coli sulfite reductase

Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an alpha(8)beta(4) quaternary structure. Protein alpha (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein beta (or sulfite reductase hemoprotein (SiR-HP)) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe(4)S(4)-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas alpha spontaneously polymerizes, we have been able to produce SiR-FP60, a monomeric but fully active truncated version of it, lacking the N-terminal part (Zeghouf, M., Fontecave, M., Macherel, D., and Covès, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the beta subunit. Pure recombinant SiR-HP behaves as a monomer in solution and is identical to the native protein in all its characteristics. Moreover, we demonstrate that the combination of SiR-FP60 and SiR-HP produces a functional 1:1 complex with tight interactions retaining about 20% of the activity of the native SiR. In addition, fully active SiR can be reconstituted by incubation of the octameric sulfite reductase flavoprotein with recombinant SiR-HP. Titration experiments and spectroscopic properties strongly suggest that the holoenzyme should be described as an alpha(8)beta(8) with equal amounts of alpha and beta subunits and that the alpha(8)beta(4) structure is probably not correct.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.     

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.