title: Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties.
abstract: 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.
authors.affiliation: Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
authors.forename: Brian J N
authors.initials: BJ
authors.lastname: Jepson
authors.forename: Lee J
authors.initials: LJ
authors.lastname: Anderson
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
authors.forename: Clare J
authors.initials: CJ
authors.lastname: Taylor
authors.forename: Clive S
authors.initials: CS
authors.lastname: Butler
authors.forename: Enrique
authors.initials: E
authors.lastname: Flores
authors.forename: Antonia
authors.initials: A
authors.lastname: Herrero
authors.forename: Julea N
authors.initials: JN
authors.lastname: Butt
authors.forename: David J
authors.initials: DJ
authors.lastname: Richardson
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D000458:Cyanobacteria / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008280:Magnetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D009566:Nitrates / Q000737:chemistry; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D011199:Potentiometry / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: D029968:Escherichia coli Proteins; D005288:Ferredoxins; D008670:Metals; D009566:Nitrates; C095754:mobA protein, E coli; D009565:Nitrate Reductases; D050901:Nitrate Reductase