FURTHER CHARACTERIZATION OF 2 DIFFERENT, REVERSIBLE ALDEHYDE OXIDOREDUCTASES FROM CLOSTRIDIUM-FORMICOACETICUM, ONE CONTAINING TUNGSTEN AND THE OTHER MOLYBDENUM

The tungsten- and the molybdenum-containing aldehyde oxidoreductases f rom Clostridium formicoaceticum show, for aldehydes, K-m values < 30 m u M and K-i values of millimolar concentrations. The tungsten-containi ng aldehyde oxidoreductase is inactivated to 50% by 3 mM KCN within 1 min, by 1 mM ferricyanide within 5 min, and by 0.05 mM chlorolhydrate within 30 s. The molybdenum-containing AOR shows 50% inactivation with in 1 min only with 70 mM KCN. The tungsten-containing enzyme is very s ensitive to oxygen, especially in the reduced state, whereas the molyb denum-containing enzyme exhibits only moderate oxygen sensitivity with out being markedly influenced by the redox state of the enzyme. The tu ngsten in the aldehyde oxidoreductase is bound to a pterin cofactor (W co) of the mononucleotide form that is known for molybdopterin cofacto r (Moco). The nature of the molybdenum cofactor in the molybdenum-cont aining aldehyde oxidoreductase is still unclear. The UV/VIS spectrum o f the tungsten-containing aldehyde oxidoreductase shows a broad absorp tion in the range of 400 nm with a millimolar absorption coefficient o f 18.1 (reduced form) and 24.8 (dehydrogenated form) at 396 nm. The ep r spectrum exhibits two different W(V) signals with the following g va lues for signal A: 2.035, 1.959, 1.899 and signal B: 2.028, 2.017, 2.0 02. Dithionite-reduced enzyme shows signals of 4Fe-4S or 2Fe-2S cluste rs. Initial rate studies with different substrates for the carboxylate reduction led to a Bi Uni Uni Bi mechanism.