US EPA

1958337 

Journal Article 

Adaptation to a High-Tungsten Environment: Pyrobaculum aerophilum Contains an Active Tungsten Nitrate Reductase 

de Vries, S; Momcilovic, M; Strampraad, MJF; Whitelegge, JP; Baghai, A; Schroeder, I 

2010 

Yes 

Biochemistry
ISSN: 0006-2960
EISSN: 1520-4995 

49 

45 

9911-9921 

English 

20863064 

10.1021/bi100974v 

WOS:000283833800028 

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 mu M 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 mu M) 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.