EPR and redox characterization of iron‐sulfur centers in nitrate reductases A and Z from Escherichia coli

Abstract

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichiu coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range + 200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are + 60 mV and + 80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4SI1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4SI1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential ironsulfur clusters of the two enzymes can be proposed.The membrane-bound complex nitrate reductase of Escherichiu coli is the terminal enzyme of the respiratory chain when the bacterium is grown anaerobically in the presence of nitrate. Previous studies have shown that it is composed of three types of subunits and that it contains three kinds of metal centers. One is a molybdenum center, which is considered to be the catalytic site; in the Mo(V) valence state, this center gives a pH-dependent EPR spectrum [l]. The variations of its amplitude as a function of the applied potential follow a bellshaped curve, which has been attributed to the interplay of Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples [2]. Secondly, there are 6-type hemes, which have so far been detected by EPR only as Fe-NO species [l]. Lastly, there are several iron-sulfur centers which were studied by EPR clusters by magnetic circular dichroism [3]. Two redox potentials were measured for these centers at 80 20 mV and 50 f 20 mV [2], but EPR spectra recorded at different redox states were interpreted in terms of the enzyme containing four or five iron-sulfur centers [3]. Therefore, the over-all stoichiometry and the redox Correspondence to B. Guigliarelli,

Characterization of the paramagnetic iron‐containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase

“…ironsulphur clusters in enzymes other than the periplasmic nitrate reductase is unknown, convincing assignment of both the high potential iron-sulphur centres of the E. coZi major membrane-bound nitrate reductase to clusters in a subunit other than MGD-binding NarG [ 19,20,48] suggests that the NarG cluster, like that of NapA, is probably of relatively low reduction potential.…”

“…Such apparently physiologically irreducible redox centres are now recognised to occur in many electron transport systems e.g. the membrane-bound nitrate and DMSO reductases [19,20,41,48] and the tetrahaem cytochrome csubtmit of the Rhodopseudomonas viridis reaction centre [49]. The function of such centres is obscure.…”

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

“…Such a very positive potential is not entirely atypical for ½4Fe4S 2þ∕1þ centers. In the MBH hydrogenases from R. eutropha H16 and R. metallidurans CH34, the reduction potential for this cluster is also very positive (28,29), while in the nitrate reductase from E. coli, both [3Fe4S] and [4Fe4S] clusters have comparable reduction potentials with those in Hase I (29). For pH values greater than 7.4 the E m of the ½4Fe4S 2þ∕1þ couple approaches a dependence of 57 mV∕pH and the proton association constant inferred is between 6.9 and 7.1 (Fig.…”

Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

“…In the absence of substrate, the form of AcnA containing [4Fe-4S]+ displays an EPR signal due to two or three species, each of which must be electronically similar. Structurally and thermodynamically similar conformers of [4Fe-4S] clusters are known to co-exist in other systems e.g., Desulphovibrio qfricanus Fd I (Hatchikian et al, 1984), E. coli membrane-bound respiratory nitrate reductases A and Z (Guigliarelli et al, 1992) and the corrinoid/(Fe-S] protein from Clostridiurn therrnoaceticuin (Ragsdale et al, 1987). The EPR parameters of the [4Fe-4S]+ signals from AcnA are also very similar to those of the signals from m-aconitases and c-aconitases, indicating that the clusters in these three aconitases are alike.…”