Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis |
| |
| Authors: | Ish K Dhawan Roopali Roy Brian P Koehler Swaranalatha Mukund Michael W W Adams Michael K Johnson |
| |
| Institution: | (1) Department of Biochemistry & Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA, GE;(2) Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA Tel.: +1-706-5429378 Fax: +1-706-5422353 e-mail: johnson@sunchem.chem.uga.edu, GE |
| |
| Abstract: | Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD)
spectroscopies. The results reveal a Fe4S4]2+,+ cluster (E
m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of
W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed
upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and
W(V)/W(VI) states at physiologically relevant potentials (E
m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists
up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples
of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox
properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential
species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E
m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E
m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total
W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential
species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for
each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts
of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible
for the catalytic activity in sulfide-activated samples of Tl FOR.
Received: 9 September 1999 / Accepted: 17 February 2000 |
| |
| Keywords: | Tungsten Aldehyde ferredoxin oxidoreductase Electron paramagnetic resonance Magnetic circular dichroism Iron-sulfur cluster |
| 本文献已被 SpringerLink 等数据库收录！ |
|
