Characterization of bacterial hydrogenase

Kidman, Antony David
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Hydrogenase, an enzyme found in microorganisms, plays an important role in the disposition of electrons during anaerobic metabolism (Grey and Gest, Science 148, 186, 1965) and has been implicated in nitrogen fixation (Lee and Wilson, J. Biol. Chem. 151, 377, 1943). Comparative studies on the physical properties have been impossible to date because of the enzyme's instability upon purification (Sadana and Jagannathan, Biochim. Biophys. Acta 19, 440, 1956). Recent investigations by Ackrell et al. using direct assay with a redox dye on acrylamide disc gels (J. Bacteriol. 92, 828, 1966) have indicated the presence of hydrogenase isoenzyrnes in a wide variety of microorganisms. In the present work three isoenzymes of C. pasteurianum (α, β and γ) were found to possess the same molecular weights in the region of 50,000. The α and β species would convert to the more stable γ species on storage overnight. Techniques such as gel electrophoresis, sucrose density gradient centrifugation, and gel filtration permitted studies on hydrogenase in the presence of other proteins by direct assay of biological activity. Kinetic studies on C. pasteurianum hydrogenase by use of the appropriate redox dyes, methyl viologen and methylene blue, showed that molybdenum was involved in one electron transfer, but not in two electron transfer. A. vinelandii hydrogenase, believed to be involved in nitrogen fixation, was subjected to time course studies to determine the stability of the isoenzymes during storage and culture growth; no significant changes in the isoenzyme band patterns were found. Hydrogenase existed in A. vinelandii in both a soluble and particulate fraction, which could be solubilized upon appropriate treatment. The molecular size (130,000) and isoelectric point (pI =5) of the soluble and solubilized preparations were found to be similar. This observation, together with the fact that soluble hydrogenase was released by the particulate material on standing and during disruption, suggested that the two preparations contained the same enzyme. Comparative studies were performed on the size and shape of the hydrogenase isoenzymes of the following organisms. (1) The strict anaerobes: C. felsineum (59,000), C. butylicum (105,000), C. butyricum (53,000), and D. desulfuricans (56,000). (2) The facultative anaerobes: P. vulgaris (185,000) and E. coli (215,000). A. vinelandii, D. desulfuricans and the facultative anaerobes which contained cytochromes possessed both soluble and particulate hydrogenase activity. The soluble material was resolved into a complex form (mol. wt. 2-3 x 10^6) and a free form. It was thought that an equilibrium existed between these two states, and furthermore that the complex was associated with the cytochromes of these organisms. Thus the size and properties of the hydrogenase enzymes showed significant differences which depended upon the type of electron transfer process occurring in the organism.
Thesis (Ph. D.)--University of Hawaii, 1968.
Bibliography: leaves [136]-141.
xiv, 141 l graphs, tables
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Theses for the degree of Doctor of Philosophy (University of Hawaii (Honolulu)). Biomedical Sciences (Biochemistry); no. 173
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