8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies.
Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.
Livingston
D J
DJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Fox
J A
JA
Orme-Johnson
W H
WH
Walsh
C T
CT
D000332:Aerobiosis; D000693:Anaerobiosis; D002248:Carbon Monoxide / Q000494:pharmacology; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*
D002248:Carbon Monoxide; D010088:Oxidoreductases; C033895:coenzyme F420 hydrogenase