Two W‐containing formate dehydrogenases (CO₂‐reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans

Abstract

Two formate dehydrogenases (CO 2 -reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionateoxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H 2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO 2 -reduction rates, and low K m values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 UAEmg )1 and a K m for formate of 0.04 mM were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 UAEmg )1 and a K m of 0.01 mM for formate with benzyl viologen as electron acceptor. The specific CO 2 -reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 UAEmg )1 and 89 UAEmg )1 , respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected.

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…W-containing enzymes generally catalyze reactions with low redox potentials ( £ )420 mV) [2,6]. In the case of W-Fdh enzymes, these were also shown to catalyze the conversion of CO 2 to formate [5], a fact not yet demonstrated in any Mo-Fdh. This is consistent with the fact that W(IV) is a stronger reducer than Mo(IV).…”

“…In the same medium where D. alaskensis Fdh was obtained, an aldehyde oxidoreductase (AOR), almost identical to the Mo-AORs from SRB, contains only Mo [53]. In contrast, D. gigas Fdh [3] and Fdh enzymes from Syntrophobacter fumaroxidans incorporate W [5], despite their growth with enough Mo in the medium. These examples suggest that bioavailability is not the only condition for an enzyme to incorporate either Mo or W and that some enzymes can function only with a specific ion, as in the case of the W-Fdh described above, the W enzymes found in hyperthermofilic archaea [2,6], and the Mo-AOR from SRB.…”

“…Evolution. Different authors seem to agree that Wcontaining isoenzymes represent, apparently, less evolved forms or ancestors of the Mo-containing enzymes [3,5,51]. In this context, the existing Wcontaining enzymes, such as D. gigas and S. fumaroxidans Fdhs [3,4], would represent a group of enzymes that cannot adopt Mo to accomplish a specific function in living organisms, which along evolution were substituting Mo for W in enzymes.…”

“…Mo-containing enzymes are found in almost all forms of life [1], whereas W-containing enzymes seem to be exclusive for organisms such as hyperthermofilic archaea that live in extreme environments [2]. However, W-containing enzymes have been found in organisms that do not need extreme conditions [3,4,5], suggesting a more important role for tungsten [6]. Some of the mononuclear W-containing enzymes described present active sites similar to the Mo enzymes, but they have different 3D structures and have been classified in a different group [2,7].…”

“…Consequently, it was suggested that the reduction of CO 2 to formate is W-dependent, whereas the inverse reaction in anaerobes is performed exclusively by Mo-containing enzymes [4]. Additional studies should be performed to verify whether the mechanism proposed for E. coli Fdh-H is valid for all formate dehydrogenases [13] and if the conversion of CO 2 to formate is exclusive for W-Fdh proteins [5].…”