Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties.
The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.
Sun
J H
JH
Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331.
Arp
D J
DJ
D000114:Acetylene / Q000494:pharmacology; D000332:Aerobiosis; D001395:Azotobacter / Q000201:enzymology*; D004227:Dithionite / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006859:Hydrogen / Q000378:metabolism / Q000494:pharmacology; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007203:Indigo Carmine / Q000494:pharmacology; D007700:Kinetics; D010100:Oxygen / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D013053:Spectrophotometry
D004227:Dithionite; D006859:Hydrogen; D007203:Indigo Carmine; D006864:Hydrogenase; D011190:Potassium Cyanide; D000114:Acetylene; D010100:Oxygen; D004229:Dithiothreitol