Tuning a Nitrate Reductase for Function

Abstract

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxindependent 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. EPRmonitored 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 membranebound 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.Nitrate is a widely used and readily available source of inorganic nitrogen for plants and microorganisms (1). Fixed inorganic nitrogen is mainly supplied to natural environments either from human agricultural or industrial activities or from biological nitrogen fixation. Most of it is converted to nitrate by nitrifying bacteria, and the nitrate then serves as a nitrogen source for assimilation or as a respiratory electron acceptor. Bacterial nitrate reductases are molybdoenzymes that can catalyze the two-electron reduction of nitrate to nitrite and can be classified into three groups according to their localization and function (2). 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) 1 cofactor and a [4Fe-4S] cluster (3, 4). These membrane-bound nitrate reductases couple quinol oxidation b...

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…Furthermore, reaction of Dd NapA with cyanide yields an EPR signal similar to those observed in the Mo-containing enzymes Pp NapAB [25], air-oxidized Nas from Cyanobacteria [29], and dithionite-reduced Fdh from Methanobacterium formicicum [36] (Table 1), which suggests similar structures for the Mo sites of these enzymes. The nitrate and cyanide signals in Dd NapA seem to be associated with hexa-and pentacoordinated Mo sites, respectively, instead of the proposed hepta-coordinated sites for the high g (nitrate) and the very high g (cyanide) signals in Pp NapAB [25,37].…”

“…A least-squares fit to the data of the FeS signal with a Nernstian function (n=1) yielded E=À390 mV (vs. NHE). This value is unusually low for Naps, which usually have FeS centers with redox potentials around À200 mV [23,25,29]. The redox potentials of the low-potential species could not be precisely determined owing to the lack of data below À500 mV (Fig.…”

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

“…15 The observations outlined have led us to propose that the electrochemical potentials over which the activities of QH 2 -dehydrogenases move between negligible and maximal values are tuned to reinforce their cellular function. 15,34 To understand why Paracoccus increases the activities of cytochrome bo 3 and NapABC in response to a lower redox poise of the UQ-pool we must consider the contribution of each QH 2 oxidising system to conserving the energy of catabolism, Fig. 4.…”

“…À0.30 V are required to see any appreciable activity from the assimilatory nitrate reductase (Nas) of Synechococcus elongatus. 34 These potentials are too low to be engaged by the Q/QH 2 -pool. However, they are ideally poised to be engaged by the low potential ferredoxin redox partner that receives electrons from photosystem I.…”

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

“…For example, in the periplasmic enzymes, cysteine provides a thiol ligand to the molybdenum ion, whereas aspartate provides one or two oxygen ligands to the molybdenum ion in the membrane-bound enzymes (3, 4, 8 -10). In evolutionary terms the periplasmic and cytoplasmic assimilatory enzymes are most closely related, and this is reflected in similar spectroscopic properties between the two enzymes, although the redox properties of the molybdenum centers have become tuned for their different metabolic functions (2,(11)(12)(13).…”