The tungsten metallome of Pyrococcus furiosus

Abstract

The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope 187 W (t 1/2 = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 mM, of which ca. 30% appears to be free tungsten, probably in the form of tungstate or polytungstates. The remaining 70% is bound by five different tungsten enzymes: formaldehyde ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase and the tungsten-containing oxidoreductases WOR4 and WOR5. The membrane proteome of P. furiosus is devoid of tungsten. The differential expression, as measured by the tungsten level, of the five soluble tungsten enzymes when the cells are subjected to a cold-shock shows a strong correlation with previously published DNA microarray analyses.

Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus

“…The intracellular free and protein-bound molybdenum and tungsten concentrations of P. furiosus were determined using catalytic-adsorptive stripping voltammetry as previously described (28). Three 100-ml serum bottles, filled with 50 ml growth medium, were used for overnight (14-h) cultivation at 95°C of P. furiosus.…”

“…After irradiation, the vial was allowed to cool to room temperature for 5 h to ease handling and to decrease the radiation from short-lived radioisotopes in the quartz vial and the aluminum irradiation tray. 2Ϫ solution was prepared as previously described (28). Protein sample preparation for MIRAGE.…”

“…The quantity of the metal inside the separate spots after native-native two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was determined using autoradiography. This method was previously used with the short-lived radioisotope 187 W to identify the W metalloproteome of P. furiosus under different growth conditions (28) and was found to compare well with transcriptomic data (32). In the present paper we describe the incorporation of Mo in enzymes of the P. furiosus AOR family using the radioisotope 99 Mo.…”

One- and two-electron reduction of molybdate reversibly bound to the archaeal tungstate/molybdate transporter WtpA

“…5 Its tungsten proteome encompasses five AORs plus intracellular free tungstate. 6 Tungsten is also transiently bound to components of the biosynthetic machinery of tungstopterin 7 and to an ABC transporter named Wtp (tungsten transport protein). The soluble oxoanion binding protein of the latter exhibits a high selectivity for tungstate (K D ª 10 -11 M) over molybdate (K D ª 10 -8 M).…”

“…8 The structural and electronic basis of this selectivity is not understood, but it appears to be related to an unusual quasi-octahedral coordination of the metal that is induced upon binding of the oxoanions to the WtpA involving one oxygen each of the carboxylate side chains of an aspartate and a glutamate residue as discovered in the crystal structure of the WtpA from Archaeoglobus fulgidus. 9 The proposal of mononuclear quasi-octahedral MoO 6 and WO 6 around the fundamental question whether such a structure would be likely, and if so, whether the formal metal oxidation state of VI would be a stable one. The quasi-octahedral coordination was recently corroborated in increased-resolution crystallographic studies on the tungstate complexes of WtpA from P. furiosus, A. fulgidus and other species, combined with EXAFS measurements on the A. fulgidus protein (Fig.…”