Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.
Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at WO(4)(2-) concentrations that are inhibitory to bacterial Nars. In this report, we establish that the enzyme purified from cells grown with 4.5 μM WO(4)(2-) contains W as the metal cofactor but is otherwise identical to the Mo-Nar previously purified from P. aerophilum grown at low WO(4)(2-) concentrations. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor. The W-Nar has a 2-fold lower turnover number (633 s(-1)) but the same K(m) value for nitrate (56 μM) as the Mo-Nar. Quinol reduction and nitrate oxidation experiments monitored by EPR with both pure W-Nar and mixed W- and Mo-Nar preparations suggest a monodentate ligation by the conserved Asp241 for W(V), while Asp241 acts as a bidentate ligand for Mo(V). Redox titrations of the Mo-Nar revealed a midpoint potential of 88 mV for Mo(V/IV). The E(m) for W(V/IV) of the purified W-Nar was estimated to be -8 mV. This relatively small difference in midpoint potential is consistent with comparable enzyme activities of W- and Mo-Nars. Unlike bacterial Nars, the P. aerophilum Nar contains a unique membrane anchor, NarM, with a single heme of the o(P) type (E(m) = 126 mV). In contrast to bacterial Nars, the P. aerophilum Nar faces the cell's exterior and, hence, does not contribute to the proton motive force. Formate is used as a physiological electron donor. This is the first description of an active W-containing Nar demonstrating the unique ability of hyperthermophiles to adapt to their high-WO(4)(2-) environment.
de Vries
Simon
S
Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Momcilovic
Milica
M
Strampraad
Marc J F
MJ
Whitelegge
Julian P
JP
Baghai
Ashkan
A
Schröder
Imke
I
D000064:Acclimatization; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004777:Environment; D007700:Kinetics; D013058:Mass Spectrometry; D050901:Nitrate Reductase / Q000302:isolation & purification / Q000378:metabolism*; D009572:Nitrite Reductases / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D041543:Pyrobaculum / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D014414:Tungsten / Q000378:metabolism / Q000494:pharmacology*
D021122:Protein Subunits; D009572:Nitrite Reductases; D050901:Nitrate Reductase; D014414:Tungsten