Electrocatalytic reduction of nitrate and selenate by NapAB.

Bacterial cellular metabolism is renowned for its metabolic diversity and adaptability. However, certain environments present particular challenges. Aerobic metabolism of highly reduced carbon substrates by soil bacteria such as Paracoccus pantotrophus presents one such challenge since it may result in excessive electron delivery to the respiratory redox chain when compared with the availability of terminal oxidant, O2. The level of a periplasmic ubiquinol-dependent nitrate reductase, NAP, is up-regulated in the presence of highly reduced carbon substrates. NAP oxidizes ubiquinol at the periplasmic face of the cytoplasmic membrane and reduces nitrate in the periplasm. Thus its activity counteracts the accumulation of excess reducing equivalents in ubiquinol, thereby maintaining the redox poise of the ubiquinone/ubiquinol pool without contributing to the protonmotive force across the cytoplasmic membrane. Although P. pantotrophus NapAB shows a high level of substrate specificity towards nitrate, the enzyme has also been reported to reduce selenate in spectrophotometric solution assays. This transaction draws on our current knowledge concerning the bacterial respiratory nitrate reductases and extends the application of PFE (protein film electrochemistry) to resolve and quantify the selenate reductase activity of NapAB.

Enhancement of photosynthetic O2 evolution in Chlorella vulgaris under high light and increased CO2 concentration as a sign of acclimation to phosphate deficiency.

The photosynthetic oxygen evolution of Chlorella vulgaris (Beijer.) cells taken from phosphate-deficient (-P) and control cultures was measured during 8 days of culture growth. Under inorganic carbon concentration (50 microM) in the measuring cell suspension and irradiance (150 micromol m(-2) s(-1)), the same as during culture growth, there were no marked differences in the photosynthetic O2 evolution rate between the -P cells and the controls. The much slower growth of -P cultures indicated that the utilization of absorbed photosynthetically active radiation (PAR) in the CO2 assimilation and biomass production were in -P cells less efficient than in the controls. Alga cells under the phosphorus stress utilized more of the absorbed PAR in the nitrate reduction than the control cells. However, under conditions of more efficient CO2 supply (inorganic carbon concentration 150 microM, introducing of exogenous carbonic anhydrase to the measuring cell suspension) and under increased irradiance (500 micromol m(-2) s(-1)), the photosynthetic O2 evolution in -P cells reached a higher rate than in the controls. The results suggest that in -P cells the restricted CO2 availability limits the total photosynthetic process. But under conditions more favorable for the CO2 uptake and under high irradiance, the -P cells may reveal a higher photosynthetic oxygen evolution rate than the controls. It is concluded that an increased potential activity of the photosynthetic light energy absorption and conversion in the C. vulgaris cells from -P cultures is a sign of acclimation to phosphorus stress by a sun-type like adaptation response of the photosynthetic apparatus.

EPR spectroscopy: a powerful technique for the structural and functional investigation of metalloproteins.

Numerous metal centers in proteins can be prepared in a redox state in which their ground state is paramagnetic. Complementary data provided by EPR, Mössbauer, electron nuclear double resonance, magnetic circular dichroism, and NMR spectroscopies have therefore played a major role in the elucidation of the structure and function of these centers. Among those techniques the most commonly used is certainly EPR spectroscopy. In this article various aspects of the current applications of EPR to the structural and functional study of metalloproteins are presented. They are illustrated by recent studies carried out in our laboratory in the field of metalloenzymes and electron transfer systems. The power of numerical simulation techniques is emphasized throughout this work.

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase.

The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.

Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii.

1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 mumol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105,000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase present in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1.998; g3 = 1.993 and with gav. = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O4(2-) bleached the e.p.r. signals which, on reoxidation after the addition of NO3(2-) to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and gav. = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent Km 998 microM) and reduced Bromophenol Blue (apparent Km 158 microM) were effective. With these donors the apparent Km values for nitrate were 70 microM and 217 microM respectively.

Isolation and partial amino acid sequence of domains of nitrate reductase from spinach.

Fragments of spinach nitrate reductase (NR) were prepared by limited proteolysis of immunopurified enzyme using both Staphylococcus aureus V8 protease and trypsin. Incubation of NR with V8 protease yielded two enzymically active fragments which could be size separated by FPLC on a Superose 12 column or subjected to further proteolysis while bound to a blue Sepharose affinity column. An NADH-ferricyanide (NADH-FR) active fragment bound to, and was eluted from, a blue Sepharose column by micromolar concentrations of NADH. A fragment with methyl viologen-NR activity was either eluted from the same column using 1 M KNO3 or on further treatment in situ on the blue Sepharose column with trypsin. Incubation of holo-NR with trypsin resulted in the loss of all terminal nitrate reducing activities but no loss in either NADH-FR activity or NADH-cytochrome c reductase activity. Two protease-sensitive regions of NR are shown which connect essentially between the flavin (FAD) and haem domains, and between the haem and molybdenum domains of NR. Amino acid analysis of the FAD- and FAD/haem-containing domains yielded two partial sequences which are compared with sequences deduced from complementary DNA (cDNA) of NR from Arabidopsis, tobacco and spinach. The deduced sequences from Arabidopsis and tobacco are found to be ca 80% and the spinach 100% homologous to the sequence obtained for spinach NR fragments.