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.
Polyakov
Konstantin M
KM
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
Boyko
Konstantin M
KM
Tikhonova
Tamara V
TV
Slutsky
Alvira
A
Antipov
Alexey N
AN
Zvyagilskaya
Renata A
RA
Popov
Alexandre N
AN
Bourenkov
Gleb P
GP
Lamzin
Victor S
VS
Popov
Vladimir O
VO
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D045263:Cytochromes a1 / Q000737:chemistry*; D003575:Cytochromes c1 / Q000737:chemistry*; D042001:Ectothiorhodospiraceae / Q000201:enzymology*; D006418:Heme / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009565:Nitrate Reductases / Q000737:chemistry*; D009573:Nitrites / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D001386:Azides; D001426:Bacterial Proteins; D009573:Nitrites; D006418:Heme; D045263:Cytochromes a1; D003575:Cytochromes c1; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome)