Isotope exchange and discrimination by the H 2-oxidizing hydrogenase from soybean root nodules

Abstract

Purified nodule hydrogenase (ferredoxin:H + oxidoreductase, EC 1.18.3.1) (in water) oxidized 2H 2 more slowly than H 2, although the effects were small. ( V)H 2/( V) 2H 2 and (V K ) H 2 ( V K ) 2 H 2 were 1.2 to 1.3 at pH 6 and 30°C. ( V)H 2/( V) 2H 2 varied with the temperature. 2H 2 evolution from 2H 2O was 2- to 3-times slower than H 2 evolution from H 2O; however, H 2 was oxidized only 5 to 10° more slowly in 2H 2O than in H 2O. An isotope exchange reaction was catalyzed by this hydrogenase. In the 2H 2-H 2O reaction, both H 2H and H 2 were formed. When the reaction was not limited by diffusion, the ratio of the rates of H 2 formation to H 2H formation was 1.5. Saturation curves of exchange activity vs. p( 2 H 2) and vs. enzyme concentration are presented. The rate of exchange activity was 5–10% of the rate of H 2 oxidation. An H 2- 2H 2O exchange also was studied; both H 2H and 2H 2 were formed.This exchange activity was 4-5-times slower than the 2H 2-H 2O reaction. The minimum ratio of the rate of 2H 2 to H 2H formation was 1.0 in 97% 2H 2O and 2.4 in greater than 99% 2H 2O. The responses of H 2 oxidation, evolution and exchange to variation in pH were determined. The isotope discrimination results indicate that dihydrogen bond breakage is not the rate-limiting step in H 2 oxidatlon. H 2 evolution and isotope exchange share the same rate-limiting step, as indicated by their similar rates, isotope effects and pH profiles.