Studies on inhibition of transformation of 2,4,6-trinitrotoluene catalyzed by Fe-only hydrogenase from Clostridium acetobutylicum

Abstract

The major enzyme in Clostridium acetobutylicum ATCC 824 leading to transformation of TNT has been reported to be the Fe-only hydrogenase. In this study, we examine the effect of inhibitors of hydrogenase on TNT reduction by Clostridial extracts. These experiments further demonstrate the major role of hydrogenase in TNT transformation. The C. acetobutylicum hydrogenase is closely related to that of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 1.18 angstroms for the Calpha atoms of the generated 3D simulation model. The Hyd1, Hyd2, and Hyd3 antibodies generated against hydrogenase reacted with both the hydrogenase in cell extracts and with C. acetobutylicum hydrogenase expressed in Escherichia coli. Inhibition studies using antibodies against Fe-only hydrogenase from C. acetobutylicum indicated that the transformation of TNT by crude cell extracts was completely inhibited by Hyd2 antibody (to amino acid 415-428) whereas antibodies Hyd1 (to residues 1-16) and Hyd3 (to amino acid 424-448) inhibited less effectively. The TNT transforming activity of the cell extract was retained when Hyd2 antibody pretreated with purified but enzymatically inactive recombinant hydrogenase was added to the extract. Addition of the transition metal Cu2+ to extracts completely inhibited the transformation of TNT suggesting the destruction of [4Fe-4S] centers which are essential for transfer of electrons from the H2-activating site to TNT. Growth of C. acetobutylicum was also inhibited by 0.5 mM Cu2+ and Hg2+ ions. The triazine dye, procion red and the nitroimidazole drug, metronidazole inhibit TNT reduction. The inhibition studies using antibodies, procion red, metronidazole, and transition metals suggest that different portions of hydrogenase are required for effective TNT reduction.

Activation and Inactivation of Hydrogenase Function and the Catalytic Cycle: Spectroelectrochemical Studies

“…EPR and UV−visible spectroscopy studies on D. gigas hydrogenase deactivated by Cu 2+ salts suggested that the loss of catalytic activity was due to the destruction of a [3Fe-4S] cluster in the electron-transfer chain . A similar deactivating effect, by Hg(II), has been reported to occur to the Ni−Fe hydrogenase from D. vulgaris . , Cu(II) and Hg(II) irreversibly inhibit the hydrogenase from T. roseopersicina. Nedoluzhko et al have reported that Pb(II) can act as electron donor to the hydrogenase but is also an irreversible inhibitor.…”

“…274 A similar deactivating effect, by Hg(II), has been reported to occur to the Ni-Fe hydrogenase from D. Vulgaris. 275,276 Cu(II) and Hg(II) irreversibly inhibit the hydrogenase from T. roseopersicina. 277 Nedoluzhko et al 278 have reported that Pb(II) can act as electron donor to the hydrogenase but is also an irreversible inhibitor.…”

“…Fe-Fe hydrogenase from D. desulfuricans was shown to be inhibited by Cu(II) but only in the presence of ascorbate. 169 The hydrogenase from D. Vulgaris 275 as well as the one involved in the reduction of 2,4,6-trinitrotoluene by hydrogen in C. acetobutylicum 276 are deactivated by Hg(II).…”

Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

“…This widely isolated species and related anaerobes are important contributors to the degradation of a variety of compounds in the oxygendepleted environment of contaminated subsurface soil. Clostridium acetobutylicum is known to enzymatically reduce TNT primarily through the activity of a Fe-only hydrogenase, which transfers electrons to TNT through an iron-sulfur center (Watrous et al 2003;Kutty and Bennett 2006;Ahmad and Hughes 2000;Hughes et al 1998b;Khan et al 1997). A more thorough understanding of the regulation of hydrogenase activity and the redox inputs to hydrogenase during TNT reduction may yield better techniques for accentuating the degradation of TNT.…”

New host-vector system in solvent-producing Clostridium saccharoperbutylacetonicum strain N1-4

“…A host-vector system in clostridia has been developed in a type strain of Acetone-Butanol fermentating clostridia C. acetobutylicum ATCC824 (Girbal et al, 2003;Kutty and Bennett, 2006;Noelling et al, 2001;Tummala et al, 2003 a,b). Since strain N1-4 is a different species from ATCC824 evolutionally (Johnson et al, 1997;Keis et al, 1995Keis et al, , 2001, the host-vector system of strain ATCC 824 cannot be applied to strain N1-4.…”