Action of Sulfhydryl Inhibitors on Different Forms of the Respiratory Chain-Linked Reduced Nicotinamide-Adenine Dinucleotide Dehydrogenase

M. Gutman, H. Mersmann, Thomas P. Singer, J. Luthy

Research output: Contribution to journalArticlepeer-review

Abstract

The reduced nicotinamide-adenine dinucleotide (NADH) dehydrogenase of the respiratory chain contains at least four, possibly five types of SH groups, modification of which leads to changes in catalytic activity. The different types of SH groups may be distinguished from each other on the basis of the conditions under which they react with mercurials and the diverse effects of mercaptide formation on the various catalytic parameters. Further, the reactivity of the different SH groups is also a function of the form in which the enzyme occuss in the preparations examined. Type I SH group reacts very rapidly with mercurials and oxidants at low concentrations with immediate and complete inactivation. It is seen only in the low (80, 000 molecular weight) form of the enzyme. Type II is seen in the high molecular weight form of the enzyme. It reacts rapidly in the cold with mercurials and oxidants but the resulting product has full catalytic activity. Phosphate prevents combination with negatively charged mercurials. The catalytically active derivative is very labile to temperature and to prolonged contact with the substrate compared with the free enzyme, so that on warming to 15-30° dehydrogenase (but not transhydrogenase) activity is irreversibly lost. Type III SH groups are seen in particulate and soluble preparations of the high molecular weight form of the enzyme. They become reactive only on reduction with substrate. Mercaptide formation with type III SH leads to loss of all catalytic activities to nearly the same extent; thus they may be located on the substrate side of the flavin in electron transport. Type IV SH groups are most readily detected in membrane-bound (particulate) preparations. Mercaptide formation is relatively fast in the cold and leads to a large increase in Km for ferricyanide, and an artifactual increase in dehydrogenase activity at Vmax. Type V SH group is detected only in particles: mercaptide formation occuss at higher mercurial concentrations and leads to loss of reactivity with the respiratory chain. Titration with mercurials under conditions where type V (and possibly IV) SH groups react results in loss of one of the two specific binding sites of piericidin A and rotenone in membrane preparations, resulting in a transition from sigmoidal to hyperbolic titration curvss of NADH oxidase activity. The interrelations of the different types of SH groups in the enzyme are examined and their possible location in the intramolecular electron transport system in the enzyme, including the possible participation of an SH ⇄ SS redox system in catalysis, are discussed.

Original languageEnglish
Pages (from-to)2678-2687
Number of pages10
JournalBiochemistry
Volume9
Issue number13
DOIs
StatePublished - 1 Jun 1970
Externally publishedYes

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