Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli

Abstract

The interconversion of nitrate reductase from Escherichia coli between low-pH and high-pH Mo(V) e.p.r. signal-giving species was re-investigated [cf. Vincent & Bray (1978) Biochem. J. 171, 639-647]. The process cannot be described by a single pK value, since the apparent pK for interconversion is raised by the presence of various anions. The low-pH form of the enzyme exists as a series of complexes with different anion ligands of molybdenum. Each complex has specific and slightly different e.p.r. parameters, but all show strong coupling of Mo(V) to a single proton, exchangeable with the solvent, having A(1H)av. 1.0 to 1.3 mT. Complexes with Cl-, F- [A(19F)av. 0.7 mT], NO3- and NO2- give particularly well-defined spectra. The high-pH form of the enzyme is now shown to bear a coupled proton. Like that in the low-pH species, this proton is exchangeable with the solvent, but the coupling is much weaker, with A(1H)av. 0.3 mT. Thus, contrary to earlier assumptions, the proton detectable by e.p.r. is probably not identical with the proton whose dissociation controls interconversion between the two species; the latter proton could be located in the protein rather than on a ligand of molybdenum. Treatment of the enzyme with trypsin [Morpeth & Boxer (1985) Biochemistry 24, 40-46] did not affect its Mo(V) e.p.r. signals.

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…The redox and EPR properties of the active site of membrane Nar obtained from different sources has been the subject of several studies [38,39,40,41,42,43]. Nar enzymes show pH-dependent Mo(V) ion EPR signals having resonances split by a solvent-exchangeable pro- Fig.…”

“…The Mo-bis-MGD, FeS clusters, and heme groups are represented as color-coded balls-and-sticks. .001, g 2 =1.986, g 3 =1.966, A av =9.3 G) and high pH (g 1 =1.987, g 2 =1.981, g 3 =1.962, A av =3.4 G) [39]. The catalytic role of these species is still in debate.…”

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

“…The redox and electron paramagnetic resonance (EPR) properties of the active site of membrane-bound nitrate reductases obtained from different sources have been the subject of several studies [9,[18][19][20]. Nars show pH-dependent Mo(V) ion EPR signals with resonances split by a solvent-exchangeable proton.…”

“…The crystal structures of NarGHI and NarGH from Escherichia coli, solved at a resolution of 1.9 Å [16] and 2.0 Å [17], respectively, showed that the catalytic NarG subunit (118-150 kDa) contains the molybdenum site and one [4Fe-4S] cluster (FS0), the NarH subunit (55-64 kDa) contains one [3Fe-4S] cluster (FS4) and three [4Fe-4S] clusters (FS1, FS2, and FS3), and the NarI subunit (19)(20)(21)(22)(23)(24)(25)(26) contains two b-type hemes (b P and b D ). All the redox cofactors are located along an electron transfer pathway (b D -b P -FS 4 -FS 3 -FS 2 -FS 1 -FS 0 -Mo) from which the nitrate receives the electrons provided by the quinol pool [15].…”

“…Nars show pH-dependent Mo(V) ion EPR signals with resonances split by a solvent-exchangeable proton. The EPR-active species are identified as ''low-pH'' (g 1 = 2.001, g 2 = 1.986, g 3 = 1.964, A av = 9.6 G) and ''high-pH'' (g 1 = 1.987, g 2 = 1.981, g 3 = 1.962, A av = 3.4 G) [19]. The catalytic significance of these two distinct Mo(V) EPR species has been intensively discussed.…”