Abstract:
The kinetic mechanism of the unidirectional
H2-oxidizing hydrogenase from soybean nodule bacteroids has
been investigated with highly purified enzyme. Measurements
of the K, for H2 vary from 0.97 to 2.6 pM, and the K,,, for
methylene blue varies from 6 to 17 pM. With H2 and
methylene blue as substrates, the initial velocity patterns are
intersecting. High levels of methylene blue are inhibitory (KI
= 2.4 mM): the inhibition is competitive vs. H2. CO is a
competitive inhibitor of H2 (KI= 157 pM) and noncompetitive
vs. methylene blue. O2 inactivates the enzyme (tl12 - 1 h) but
also is a reversible inhibitor of hydrogenase activity upon short
exposure to low concentrations. Inhibition by O2 is uncom-petitive vs. H2 and noncompetitive vs. methylene blue. Hydrogenase
was not inhibited by C2H2; preincubation under C2H2
inactivates the enzyme. Reduced methyl and benzyl viologens
support low rates of H2 evolution by the hydrogenase. The
K, for reduced methyl viologen is 11 pM. H2 is a potent
inhibitor of H2 evolution: the inhibition is noncompetitive vs.
reduced methyl viologen. The hydrogenase will catalyze a low
rate of exchange in the reaction between D2 and H20 to form
both HD and H2. We p r o p a two-site ping-pong mechanism
for the enzyme in which H2 is reversibly activated at one site
and e- carriers interact at the second site.
