title: Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.
abstract: 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.
authors.affiliation: Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
authors.forename: Milica
authors.initials: M
authors.lastname: Momcilovic
authors.forename: Marc J F
authors.initials: MJ
authors.lastname: Strampraad
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Ashkan
authors.initials: A
authors.lastname: Baghai
authors.forename: Imke
authors.initials: I
authors.lastname: Schröder
mesh_terms: 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*
chemical_list: D021122:Protein Subunits; D009572:Nitrite Reductases; D050901:Nitrate Reductase; D014414:Tungsten