Mo(V) Electron Paramagnetic Resonance Signals from the Periplasmic Nitrate Reductase of Thiosphaera Pantotropha

Abstract

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphueru pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrifcans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of 7: pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.Molybdenum-containing enzymes are found in all forms of life from bacteria to higher plants and man (Coughlan, 1980). With the exception of the molybdenum-containing nitrogenases which contain a novel molybdenum-iron-sulphur cluster (Burgess, 1990;Kim and Rees, 1992), the molybdenum-containing enzymes all contain one of a series of pterin molybdenum cofactors (Rajagopalan and Johnson, 1992). Enzymes of this latter type, the oxomolybdenum enzymes, participate in a wide variety of biologically important processes including nitrate assimilation in plants and microorganisms, many modes of bacterial respiration and, in higher animals, purine catabolism and sulphite oxidation. Largely on the basis of their spectroscopic properties, Wootton et al. (1991) classified the oxomolybdenum enzymes into the families of molybdenum hydroxylases, sulphite oxidases, plant and fungal assimilatory nitrate reductases and bacterial respiratory reductases. The latter class includes the respiratory nitrate reductases and dimethylsulphoxide reductases, though recent work (Bennett et al., 1994a,b) has highlighted significant differences within this group.Three types of molybdenum-containing nitrate reductases are found in bacteria ; the membrane-bound and periplasmic respiratory enzymes and the assimilatory enzyme. The membrane-bound respiratory enzymes are used by cells to utilise nitrate as the terminal electron acceptor in the absence of Correspondence to B. Bennett, School of Chemical Sciences, University of East Anglia, University Plain, Nonvich, England NR4 7TJAbbreviations. ENDOR, electron-nuclear double resonance ; EX-AFS, extended X-ray absorption fine structure; MCD, magnetic circular dichroism; pD,, , apparent pD (p*H).oxygen. The periplasmic respiratory enzymes c...

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

“…The High g Resting signal of P. pantotropus NapAB (Fig. 3A and Table 3) is present in as-isolated air-oxidized enzyme, where it can account for up to 20% of the total molybdenum ions in the sample (8,41). We also note that the Mo 5ϩ signal present in as-isolated R. sphaeroides NapAB has very similar features (Table 3) (10).…”

“…It is also notable that the E. coli Nap Mo 5ϩ signal is very similar to another High g signal of P. pantotrophus NapAB that was also originally generated in the presence of dithionite and nitrate and therefore termed High g Nitrate ( Fig. 2 and Table 3) (10,41). The possibility has been raised previously that this signal was catalytically relevant, and the High g Resting signal was not.…”

“…ied in some detail for the NapAB enzyme complex from P. pantotrophus and for which a number of Mo 5ϩ EPR signals have been reported that must reflect some plasticity of the active site pocket in this redox state (8,41 (8). The signal detected in E. coli NapA falls into the High g group, and no evidence of any of the Low g signals was apparent.…”

Tuning a Nitrate Reductase for Function

“…The Mo(V) EPR signals identified in NarB also have strong similarity to signals detected in the P. pantotrophus periplasmic nitrate reductase and A. vinelandii assimilatory nitrate reductase and termed very high g and high g on the basis of the g av values (8,16,30,34). The very high g signals are most likely to arise from inactive forms of the enzyme.…”

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

“…The nitrate signal resembles those designated as high g signals in Pp NapAB (g values in Table 1) [25,35], which were assumed to be from a catalytic intermediate. 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.…”