Alexey N. Antipov

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

New enzyme belonging to the family of molybdenum-free nitrate reductases.

A novel molybdenum-free nitrate reductase was isolated from the obligate chemolithoautotrophic and facultative anaerobic, (halo)alkaliphilic sulphur-oxidizing bacterium Thioalkalivibrio nitratireducens strain ALEN 2. The enzyme was found to contain vanadium and haem c as cofactors. Its native molecular mass was determined as 195 kDa, and the enzyme consists of four identical subunits with apparent molecular masses of 57 kDa. Apart from nitrate, the enzyme can utilize nitrite, chlorate, bromate, selenate and sulphite as electron acceptors. Moreover, it also has a haloperoxidase activity.

Molybdenum-free nitrate reductases from vanadate-reducing bacteria

Two catalytically distinct molybdenum‐free dissimilatory nitrate reductases, a soluble periplasmic and a membrane‐bound one, were isolated from the vanadate‐reducing facultatively anaerobic bacterium Pseudomonas isachenkovii and purified to electrophoretic homogeneity. The enzymes did not contain molybdenum, the periplasmic enzyme contained vanadium, whereas the membrane‐bound enzyme was vanadium‐free. Both nitrate reductases lacked molybdenum cofactor. This fact was proved by reconstitution of the apoprotein of the nitrate reductase of Neurospora crassa nit‐1 mutant. This is the first demonstration of molybdenum‐free and molybdenum cofactor‐free nitrate reductases.

Vanadium-binding protein excreted by vanadate-reducing bacteria.

A vanadium‐binding protein was isolated from the culture medium of the vanadium‐reducing bacterium Pseudomonas isachenkovii by utilizing vanadate as the terminal electron acceptor upon anaerobic respiration. The protein was associated with vanadium at a molar ratio of approximately 1:20. It was purified to homogeneity and separated into three components by treatment with 1 M HCl followed by gel filtration: a protein, a vanadium‐binding ligand, and inorganic vanadium. Electron paramagnetic resonance analysis showed that vanadium was associated with the protein in the 4+ oxidation state. The distribution of vanadium within the cell was studied by electron microscopy and x‐ray microanalysis of P. isachenkovii cells. The results suggest that vanadium, accumulated in special swells on the surface of the cell membranes, is reduced and excreted to the medium.

Halomonas campisalis , an Obligatorily Alkaliphilic, Nitrous Oxide-Reducing Denitrifier with a Molybdenum Cofactor-Lacking Nitrate Reductase

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1–68.1 mol %) and DNA–DNA homology (75–92%) and represent different morphotypes of the same species. On the basis of results of partial 16S rRNA sequencing, strain Z-7398-2 was found to be most closely related to the Halomonas campisalis isolate from the Alkali Lake (USA). The DNA–DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide–reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a molybdenum cofactor.

Vanadate Reduction by Molybdenum‐Free Dissimilatory Nitrate Reductases from Vanadate‐Reducing Bacteria

Molybdenum‐ and molybdenum cofactor‐free nitrate reductases recently isolated by us from vanadate‐reducing bacteria Pseudomonas isachenkovii are likely to mediate vanadate reduction. During anaerobic growth of P. isachenkovii on medium supplemented with nitrate and vanadate, vanadate dissimilation was followed by nitrate consumption, and this process was associated with some structural reorganizations of nitrate reductases. The homogeneous membrane‐bound nitrate reductase of P. isachenkovii reduced vanadate with NADH as an electron donor.

Characterization of Molybdenum-Free Nitrate Reductase from Haloalkalophilic Bacterium Halomonas sp. Strain AGJ 1-3

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. Strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130–140 kD monomeric protein with specific activity of 250 µmol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70–80°C.

Vanadate reduction under alkaline conditions by haloalkaliphilic Halomonas strains

Growth in the presence of vanadate and dissimilatory vanadate reduction under alkaline conditions were shown for a number of haloalkaliphilic Halomonas strains. Vanadate, which contains five-valent vanadium, was reduced to four- or three-valent compounds. Nitrate reductase plays the key role in vanadate reduction under alkaline conditions. The compounds containing reduced vanadium were obtained in crystalline form.

Isolation and preliminary characterization of new cytochrome c from autotrophic haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

New small cytochrome c (TniCYT) was purified from haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. The protein was analyzed by mass spectrometry as well as using visible, CD and EPR spectroscopy. It was found that TniCYT is a monomer with a molecular mass of 9461 Da which contains two hemes per molecule. The data of CD and EPR spectroscopy showed that two hemes possess different optical activity and are in distinct, high and low spin states. TniCYT was also demonstrated to have unusual characteristics in the visible spectrum, namely, the splitting of characteristic peaks was observed in α- and β-bands of the heme spectrum when the reduced form of cytochrome was analyzed. The α-band has two peaks with maximum at 548 and 556 nm whereas the β-band showes ones at 520 and 528 nm. According to the MALDI finger-print analysis, the new cytochrome has a unique amino acid sequence.