The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli[Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol.178, 6736–6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues‐Pousada, C. (1999) Eur. J. Biochem. 259, 235–243]. Thus, the SOD activity of Dfx isolated from the sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U·mg−1, which increases approximately 2.5‐fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low‐spin iron species with g‐values at 2.27 (g⊥) and 1.96 (g∥). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs.  Evidence for another SOD in D. desulfuricans is also presented – this enzyme is inhibited by cyanide, and N‐terminal sequence data strongly indicates that it is an analogue to Cu,Zn‐SODs isolated from other sources. This is the first indication that a Cu‐containing protein may be present in a sulphate‐reducing bacterium.

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The hybrid cluster proteins from the sulfate reducing bacteria Desulfovibrio desulfuricans ATCC 27774 ( Dd) and Desulfovibrio vulgaris strain Hildenborough ( Dv) have been isolated and crystallized anaerobically. In each case, the protein has been reduced with dithionite and the crystal structure of the reduced form elucidated using X-ray synchrotron radiation techniques at 1.25 A and 1.55 A resolution for Dd and Dv, respectively. Although the overall structures of the proteins are unchanged upon reduction, there are significant changes at the hybrid cluster centres. These include significant movements in the position of the iron atom linked to the persulfide moiety in the oxidized as-isolated proteins and the sulfur atom of the persulfide itself. The nature of these changes is described and the implications with respect to the function of hybrid cluster proteins are discussed.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.

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Neelaredoxin, a small non‐heme blue iron protein from the sulfate‐reducing bacterium Desulfovibrio gigas[Chen, L., Sharma, P., LeGall, J., Mariano, A.M., Teixeira M. and Xavier, A.V. (1994) Eur. J. Biochem.226, 613–618] is shown to be encoded by a polycistronic unit which contains two additional open reading frames (ORF‐1 and ORF‐2) coding for chemotaxis‐like proteins. ORF‐1 has domains highly homologous with those structurally and functionally important in methyl‐accepting chemotaxis proteins, including two putative transmembrane helices, potential methylation sites and the interaction domain with CheW proteins. Interestingly, ORF‐2 encodes a protein having homologies with CheW proteins.  Neelaredoxin is also shown to have significant superoxide dismutase activity (1200 U·mg–1), making it a novel type of iron superoxide dismutase. Analysis of genomic data shows that neelaredoxin‐like putative polypeptides are present in strict anaerobic archaea, suggesting that this is a primordial superoxide dismutase.  The three proteins encoded in this operon may be involved in the oxygen‐sensing mechanisms of this anaerobic bacterium, indicating a possible transcriptional mechanism to sense and respond to potential stress agents.

The structures of the hybrid cluster proteins (HCPs) from the sulfate-reducing bacteria Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (Hildenborough) have been elucidated at a resolution of 1.25 A using X-ray synchrotron radiation techniques. In the case of the D. desulfuricans protein, protein isolation, purification, crystallization and X-ray data collection were carried out under strict anaerobic conditions, whereas for the D. vulgaris protein the conditions were aerobic. However, both structures are essentially the same, comprising three domains and two iron-sulfur centres. One of these centres situated near the exterior of the molecules in domain 1 is a cubane [4Fe-4S] cluster, whereas the other, located at the interface of the three domains, contains the unusual four-iron cluster initially found in the D. vulgaris protein. Details of the structures and the associated EPR spectroscopy of the D. desulfuricans protein are reported herein. These structures show that the nature of the hybrid cluster, containing both oxygen and sulfur bridges, is independent of the presence of oxygen in the isolation and crystallization procedure and also does not vary significantly with changes in the oxidation state. The structures and amino acid sequences of the HCP are compared with the recently elucidated structure of the catalytic subunit of a carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans and related dehydrogenases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0326-y.