The stereospecificity of nitrate reductase for hydrogen removal from reduced pyridine nucleotides.

The stereospecificity of the hydrogen removal from reduced pyridine nucleotides catalyzed by nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1, and NAD(P)H : nitrate oxidoreductase, EC 1.6.6.2) was investigated. A high degree of enzyme purification was required to obtain conclusive results. Improvements are described for the purification of nitrate reductase from Chlorella fusca and from spinach (Spinacea oleracea, L.) leaves. The latter enzyme is shown to contain a cytochrome. With highly purified nitrate reductase preparations from Cl. fusca, Neurospora crassa, Rhodotorula glutinis and spinach leaves the stereospecificity of the reaction was determined to be predominantly of the A-type in all cases.     

Kinetic and mechanistic studies on the reduction of melilotate hydroxylase by reduced pyridine nucleotides.

The reduction of the melilotate hydroxylase . 2-OH-phenyl propionate complex by NADH and reduced 3-acetyl pyridine adenine dinucleotide (AcPyNADH) has been investigated using steady state kinetic and rapid reaction techniques. Reduction by NADH appeared to involve only one charge-transfer-type intermediate (between reduced enzyme and NAD) as previously described (Strickland, S., and Massey, V. (1973) J. Biol. Chem. 248, 2953-2962). Reduction by AcPyNADH was shown to involve two charge-transfer-type intermediates. The first was between oxidized enzyme and AcPyNADH and the second was between reduced enzyme and AcPyNAD. Reaction of AcPyNADH with oxidized enzyme . 2-OH-phenyl propionate complex to form the first charge-transfer complex reached equilibrium within the mixing time of the stopped flow apparatus (5 ms). Subsequent steps in the reaction appeared to be first order and were independent of the AcPyNADH concentration. An 8-fold deuterium isotope effect on the step involving flavin reduction was found when reduced 3-acetyl[4A-2H]pyridine adenine dinucleotide (AcPyNADD) was used as the reductant. Analysis of the rapid reaction results for the reaction of oxidized pyridine nucleotide with reduced enzyme . 2-OH-phenyl propionate complex indicated the presence of two forms of reduced enzyme (in equilibrium) of which only one form was capable of reacting with the oxidized pyridine nucleotide. Based on the rapid reaction data, a mechanism for the reduction half-reaction is proposed. The turnover number calculated from this mechanism is in good agreement with that determined from the steady state data.     

Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl

Highly purified plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis or by a two-phase polymer separation system oxidize reduced pyridine nucleotides, NADH or NADPH, at rates of 2-5 nanomoles/mg protein/min. These rates are not influenced by mitochondrial inhibitors or by inhibitors of the alternate respiratory pathway. The NADH oxidase has a Km of 200 microM NADH. The enzyme activity is stimulated by Ca2+ and Mg2+ ions. The function of this enzyme is unknown at present, but it may represent a redox-controlled proton pump linked to acidification.     

The oxidation and reduction of pyridine nucleotides by Rhodopseudomonas spheroides and Chlorobium thiosulfatophilum

Summary The light-induced formation of NADH by whole cells of Rhodopseudomonas spheroides has been followed fluorimetrically and found to lag slightly behind cytochrome c oxidation. The uncoupler, FCCP¹, abolished NADH formation which was also inhibited by HOQNO¹. Electron flow from NADH to oxygen or cytochrome c was inhibited in chromatophores of R. spheroides by HOQNO, antimycin A and rotenone. From the known properties of the inhibitors used it is deduced that NADH formation in the light is dependent upon reversed electron flow. No light-induced formation of NAD(P)H by whole cells or chromatophores of Chlorobium thiosulfatophilum was detected either fluorimetrically or by extraction followed by enzymic assay although cytochrome c oxidation was extensive in whole cells. Extracts of C. thiosulfatophilum catalysed the rapid reduction of endogenous or mammalian cytochrome c; unlike R. spheroides this activity was found almost entirely in the soluble fraction and was insensitive to HOQNO, antimycin A and rotenone. No cytochrome b was detected in C. thiosulfatophilum by difference spectroscopy of pyridine haemochromes of acetone powders. The K _m for NADH of NADH-cytochrome c reductase in both organisms was about 3 mol; the reductase was inhibited by NAD. The rates of NADPH-cytochrome c reductase in R. spheroides particles were too low for K _m determination; for C. thiosulfatophilum particles the K _m for NADPH was about 300 mol. The addition of NADH to soluble extracts of either organism caused the reduction of endogenous flavin that was reoxidised by ferricyanide. The NADH-cytochrome c reductase of C. thiosulfatophilum was not separated from ferredoxin on a DEAE column. It is concluded that in C. thiosulfatophilum the formation of NADH in an energy-linked reaction is unlikely; the possibility of a cyclic electron flow involving chlorophyll, ferredoxin, flavoprotein and cytochrome c is discussed.     

The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.     

Hysteretic behavior of nitrate reductase. Evidence of an allosteric binding site for reduced pyridine nucleotides.

In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.