A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus

Abstract

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe-4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80°C and pre-steady state conditions at 50°C, in the latter case via monitoring of the relatively weak (e & 2 mM -1 cm -1 ) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K M value for free formaldehyde (21 lM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K M of ferredoxin (14 lM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k obs1 = 4.7 s -1 , k obs2 = 1.9 s -1 ) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron-sulfur center in the absence of an external electron acceptor are slower (k obs3 = 6.10 · 10 -2 s -1 , k obs4 = 2.18 · 10 -2 s -1 ).On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.

Tungsten-dependent formaldehyde ferredoxin oxidoreductase: Reaction mechanism from quantum chemical calculations

“…The resulting model consists of 104 atoms. The oxidation state of W is +6, which is believed to be the reactive state [24], and thus the total charge of this model is − 1. …”

“…The redox properties of the tungsten center in AORs from P. furiosus and Pyrococcus strain ES-4, and FORs from Thermococcus litoralis and P. furiosus have been investigated by EPR redox titrations [19][20][21][22][23][24]. A redox cycle between W IV /W V and W V /W VI has been characterized through EPR spectrum of W V , which has a doublet electronic configuration.…”

“…Furthermore, the formaldehyde substrate was proposed to be oxidized by W VI with a two-electron transfer to the metal center affording W IV . The two electrons are then transferred to the ferredoxin cofactors, regenerating the W VI species [24].…”

CHAPTER 1. Molybdenum and Tungsten-Containing Enzymes: An Overview

“…516 b) Contrary to the reaction mechanisms of Xo, so or dMsor, which are presently well understood, the tungstoenzymes were less thoroughly studied; still, there are several recent remarkable breakthroughs. 508,511,514,516,[518][519][520] on the other hand, the formate oxidation mechanism is believed to be, overall, similar to the molybdenum-containing enzymes.…”

Comparative Study of the Antimicrobial Effects of Tungsten Nanoparticles and Tungsten Nanocomposite Fibres on Hospital Acquired Bacterial and Viral Pathogens

“…This oxyanion has similarities to vanadate and molybdenum and studies have shown tungsten to hold a biological function in some prokaryotes. In these organisms, tungstoenzymes, such as formate dehydrogenase, formyl methanufuran dehydrogenase, acetylene hydratase and a class of genetically related oxidoreductases, use tungsten as a tungsten-pterin to catalyse the reversible reduction of carboxylic acids to aldehydes [ 23 , 24 ]. The exploration of tungsten’s antiviral and antibacterial properties has stemmed from the Ren et al patent [ 25 ] claiming the viricidal efficacy of tungsten nanoparticles, especially in combinations with other effective antimicrobial components.…”