In vitro requirements for formate dehydrogenase activity from Desulfovibrio

Abstract

I RIEDGRER-HENDERSON, M. A., and H. D. PECK. JR. 1986. In vitro requirements for formate dehydrogenase activity from Desulfnvibrio. In DesuIfovibrio the proteh(s) involved in formate dehydrogenase activity have not been identified or characterized. In situ assays in polyacrylamide gels demonstrated that formate dehydrogenase from either D. gigas or D . vulgaris catalyzed the direct reduction of either methylene blue or benzyl viologen in the presence of formate. Thus, the same protein was active with either electron acceptor. Although the enzyme could be stored in air without irreversible inactivation by 0 2 , activity with either dye was stimulated by the addition of thiols to the arsay mixture. In the absence of formate the thiols served as a substrate for the in sirtr reduction of methylene blue or benzyl viotogen by the enzyme. Ammonium sulfate fractionation revealed the presence of a fraction which selectively stimulated activity with either benzyl viologen or cytochrome c3 as the electron acceptor. The stimulating fraction was nondialyzable, heat labile, and unstable upon storage. The fraction from either species could stimulate the fomate dehydrogenase activity of the other species. The protein may be of physiological signficance as it increased when the cells were grown on forrnate, and it stimulated the formate hydrogenlyase system with cytochrome c3 as the electron carrier. R~EDERER-HENDERSON, M. A.. et H. D. PECK, JR. 1986. In vitro requirements for formate dehydrogenase activity from DesulJovibrio. Can. J. Microbiol. 32: 425-429.Chez Desulfovibrio, la (ou les) protCine(s) impliqute(s) dans I'activitC de la formate dCshydrogCnase n'a (ou n'ont) pas Ct C identifiCe(s1 ou caracteride(s). Des essais in situ sur gels polyacrylamides ont dCmontrk que la formate dCshydrogCnase, soil de D. gigas ou de D. vulgaris, catalysait directement la kduction du bleu de mCthyl&ne ou du viologene de benzyle en prksence du formate. C'est ainsi que la m&me proteine fut active avec I'un ou I'autre des accepteurs dlClectrons. Bien que I'enzyme pouvait Stre entreposk en presence d'air sans inactivation irr6versible par 1'02, I'activitC avec I'un ou I'autre des colorants fut stimulCe par I'addition de thiols aux melanges test&. En absence de formate, les thiols ont semi de substrat a l'enzyme pour la reduction in slru du bIeu de mkthylbne et du viologene de knzyle. Une fractionation au sulfate d'ammonium a rev616 la prCsence d'une fraction qui stimulait sklectivement I'activit6. soit avec le viologene de benzyle, soit avec le cytochrome c3, comme accepteurs d'tlcctrons. Lafracrton stirnulante s'esz avedc non dialysable, labile a la chaleur et instable en entreposage. Une fraction de I'une ou de I'autre espi.ce pouvait stirnuler I'activid de la formate deshydrogknase chez I'autre espkce. La protCine peut avoir une signification physiologique, en ce qu'elle s'accroit lorsque Ics cellules sont mises B croitre sur du formate et elle stimule le syszkme formale hydrogene-lyase avec Ie cytochrome c-, comme transporteur d'Clectrons.[...

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase

“…Most FDH enzymes characterized so far are described to be inactivated in the presence of O 2 (referred to as oxygen-sensitive enzymes). − To prevent damage by O 2 and to stabilize the enzyme, inhibitors like azide (N 3 – ) or nitrate (NO 3 – ) are added during the purification of the enzyme, inhibitors that are known to significantly increase the stability of most FDH enzymes . FDHs that exhibit activity in the presence of O 2 with inhibitor present (referred to as oxygen-tolerant enzymes) were described for the Mo-containing enzymes from R. capsulatus and C. necator and the W-containing enzyme from D. vulgaris Hildenborough. ,,, What contributes to the higher stability of some enzymes (e.g., from various Desulfovibrio specimen ,− and Methylobacterium extorquens , ) in the presence of O 2 without inhibitor present, while other enzymes are extremely sensitive to the exposure of O 2 (e.g., E. coli FdhF) is not known so far. While hydrogenases act as oxygenases reducing O 2 to reactive oxygen species (Section ) or water (Section ), the O 2 sensitivity of FDH seems to follow other molecular principles.…”

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe-4S] Clusters

“…Although DCPIP data is not available for the latter enzyme, a close ortholog (Dg-FDH1) only shows 10% of BV activity with the high-potential acceptor. 18 Such linkages take on special significance when multiple FDHs encoded by the same organism are compared. In 1957, Peck and Gest 80 discovered two types of FDH in Escherichia coli solely based on their preference for artificial electron acceptors -one was linked to PMS/DCPIP and its expression was confined to O 2 /nitrate-respiring cells while the other was BV-linked and unique to nonrespiring cells (reviewed by Stewart 39 ).…”

“…Despite the paucity of information regarding redox partners (two well characterized systems exhibit low reduction potentials. 18,46 ), our central hypothesis was inspired by Yagi's 1969 observation that an FDH from D. vulgaris Miyazaki (DvM) preferentially transfers electrons to a highpotential cytochrome c 553 . 47,48 Because the genetically tractable DvH 49,50 is closely related to DvM 51 and encodes a 73% identical cytochrome c 553 (E m,7 = +62 mV), 52 we probed the O 2 sensitivity of periplasmic FDHs from this SRB shown to thrive in microaerobic niches.…”

“…For example, DvH-FDH3 has been reproducibly shown to be O 2 sensitive 14,15 while its ortholog from D. desulfuricans ATCC 27774 (Dd) can be purified in air. 16,17 Similarly, D. gigas (Dg) FDH1 is readily isolated and stored under atmospheric conditions 18,19 but its counterpart from DvH requires the presence of 10 mM sodium nitrate to prevent O 2 inactivation. 20 Regardless of the purification protocols, the resulting enzymes are 'dead on arrival' in that they must be resurrected by lengthy incubations with high concentrations of thiols (10 -50 mM dithiothreitol 20 for DvH-FDH1 and 130 mM βmercaptoethanol 16,17,19 for Dd-FDH3 and Dg-FDH1) and/or formate 17,21 prior to catalytic measurements under anaerobic conditions (a representative example can be found in Figure S5a of Oliveira et al 20 ).…”

“…A satisfactory molecular explanation for these phenomenological observations has not been forthcoming for over three decades. 18 The situation is equally unclear with FDHs isolated from organisms other than sulfate-reducing bacteria (SRB). Escherichia coli Fdh-H has been purified and characterized in the presence of sodium azide to minimize O 2 inactivation.…”