Tuning a Nitrate Reductase for Function: THE FIRST SPECTROPOTENTIOMETRIC CHARACTERIZATION OF A BACTERIAL ASSIMILATORY NITRATE REDUCTASE REVEALS NOVEL REDOX PROPERTIES

Brian J.N Jepson,Lee J Anderson,Luis M Rubio,Clare J Taylor,Clive S Butler,Enrique Flores,Antonia Herrero,Julea N Butt,David J Richardson

doi:10.1074/jbc.m402669200

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.

Highlights

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases
The synthesis of assimilatory nitrate reductases in bacteria is insensitive to aerobiosis but inhibited by ammonium, contrasting with both the respiratory nitrate reductase groups [11, 12]
Transport is followed by the two-electron reduction of nitrate to nitrite catalyzed by the assimilatory nitrate reductase and the further six-electron reduction of nitrite to ammonium by a sirohemecontaining nitrite reductase

Summary

Introduction

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. Respiratory membrane-bound nitrate reductases are generally integral membrane protein complexes with the active site located on the cytoplasmic face of the cytoplasmic membrane and are constituted by subunits (e.g. NarI and NarH) that mediate electron transfer from the quinol pool to the catalytic subunit, NarG, which contains a bismolybdopterin guanine dinucleotide (bis-Mo-MGD) cofactor and a [4Fe-4S] cluster [3, 4]. These membrane-bound nitrate reductases couple quinol oxidation by nitrate to the generation of a transmembrane proton electrochemical gradient, and their synthesis is normally unaffected by ammonium but repressed by oxygen [2]. The ammonium is incorporated into carbon skeletons, mainly utilizing the glutamine synthetase (GS)/glutamine:2-oxoglutarate amidotransferase (GOGAT) pathway (12, 18 –20)

Methods

Results

Conclusion