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,
The Desulfovibrio gigas hydrogenase is a typical (NiFe) hydrogenase containing a Ni center and three FeS centers, one [3Fe-4S] and two [4Fe-4S] clusters. When the enzyme is activated under hydrogen gas, the Ni center becomes paramagnetic, giving a characteristic electron paramagnetic resonance (EPR) signal with g values at 2.19, 2.14 and 2.01, the Ni-C signal. Two redox states of the enzyme can be prepared, in which the [4Fe-4S] clusters are either diamagnetic or paramagnetic. In this latter state, the magnetic coupling between metal centers induces both the appearance at low temperature of a complex EPR spectrum, the split Ni-C signal, and a significant enhancement of the relaxation rates of the Ni center. Good simulations of the split Ni-C signal recorded at three different microwave frequencies (X-band, Q-band, and S-band) are obtained by using a model based on a point dipole approximation of the dipolar and exchange interactions between paramagnets. The spectral analysis demonstrates that only one [4Fe-4S]1+ cluster is significantly coupled to the Ni site and provides a detailed description of the relative arrangement of the two centers. In addition, the magnetic characteristics of this [4Fe-4S]1+ cluster can be deduced from the simulations. Moreover, the spin-spin and spin-lattice relaxation times of the interacting centers were measured in the two redox states of the enzyme, either by power saturation and pulsed EPR experiments at low temperature or from the broadening of the EPR lines at higher temperature. The relaxation behavior of the Ni center is well explained by using in the theoretical analysis, the set of structural and magnetic parameters deduced from the spectral simulations. Our structural conclusions on the active D. gigas hydrogenase are compared to the preliminary data of a low-resolution crystal structure of the oxidized enzyme [Volbeda, A., Piras, C., Charon, M. H., Hatchikian, E. C., Frey, M., & Fontecilla-Camps, J. C. (1993) News Lett. Protein Crystallogr. 28, 30-33].
The structural organization of paramagnetic centers in biomolecules can be predicted on the basis of a quantitative study of their magnetic interactions. These studies are usually carried out within the framework of the so-called point dipole approximation, in which the delocalization of the magnetic moments over the centers is ignored. In this paper, we examine how this delocalization can be taken into account in the spin Hamiltonian describing the magnetic interactions between polynuclear paramagnetic centers. A local spin model is described and applied first to a system made of a dinuclear center interacting with a mononuclear center and then to a system comprising two dinuclear centers. In both cases, the EPR spectrum calculated from the local spin model is compared to that given by the point dipole model for different geometrical configurations. The model is illustrated by a detailed quantitative study of the magnetic interactions between the molybdenum center and one [2Fe-2S] center (center 1) in the enzyme xanthine oxidase. These studies emphasize the effective character of some important structural parameters given by numerical simulations of EPR spectra based on the point dipole approximation.
In typical NiFe hydrogenases like that from Desulfovibrio gigas, the active state of the enzyme which is obtained by incubation under hydrogen gas gives a characteristic Ni-C electron paramagnetic resonance (EPR) signal at g = 2.19, 2.14, and 2.01. The Ni-C species is light-sensitive, being converted upon illumination at temperatures below 100 K in a mixture of different Ni-L species, the most important giving an EPR signal at g = 2.30, 2.12, and 2.05. This photoprocess is considered to correspond to the dissociation of a hydrogen species initially coordinated to the Ni ion in the Ni-C state. When the [4Fe-4S] centers of the enzyme are reduced, the proximal [4Fe-4S]1+ cluster interacts magnetically with the Ni center, which leads to complex split Ni-C or split Ni-L EPR spectra only detectable below 10 K. In order to probe the structural changes induced in the Ni center environment by the photoprocess, these spin-spin interactions were analyzed in D. gigas hydrogenase by simulating the split Ni-L spectra recorded at different microwave frequencies. We shown that, upon illumination, the relative arrangement of the Ni and [4Fe-4S] centers is not modified but that the exchange interaction between them is completely canceled. Moreover, the rotations undergone by the Ni center magnetic axes in the photoconversion were determined. Taken together, our results support a Ni-C structure in which the hydrogen species is not in the first coordination sphere of the Ni ion but is more likely bound to a sulfur atom of a terminal cysteine ligand of the Ni center.
The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single [3Fe-4S] center and the three [4Fe-4S] centers. The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. With small variations, the properties of these mutant enzymes do not differ from one another. They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present. Although physiological activity was reduced, it was sufficient to allow growth on nitrate. The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme. Spectral EPR analysis showed that the [4Fe-4S] center of high redox potential (center 1, +80 mV) was missing. The loss of this center did not affect the stable integration of the other three centers. The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press). Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the [4Fe-4S] center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable. On the basis of these results, it is proposed that the [3Fe-4S] center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.
In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.