A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus.
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 degrees C and pre-steady state conditions at 50 degrees C, in the latter case via monitoring of the relatively weak (epsilon approximately 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 KM value for free formaldehyde (21 microM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 microM) 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 (kobs1 = 4.7 s(-1), kobs2 = 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 (kobs3 = 6.10 x 10(-2 )s(-1), kobs4 = 2.18 x 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.
Bol
Emile
E
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
Broers
Nicolette J
NJ
Hagen
Wilfred R
WR
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000445:Aldehyde Oxidoreductases; C080011:formaldehyde ferredoxin oxidoreductase