Pedro Tavares

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2 Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S–>Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Denitrification, or dissimilative nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. In this manuscript we present a concise updated review of the bioinorganic aspects of denitrification. (c) 2006 Elsevier Inc. All rights reserved.

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe-B). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while FeB was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g similar to 6 and g similar to 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g similar to 6 region were assigned to a small quantity of uncoupled high-spin Fe-III heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin spin coupled binuclear center comprising the low-spin Fe-III heme b(3) and the high-spin Fe-B(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe-II-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe-B(II) site. The function of heme b(3) would then be to orient the Fe-B-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.