The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Differences between the Reactivities of Two Pyridine Nucleotides in the Rapid Reduction Process and the Reoxidation Process of Adrenodoxin Reductase
Toshihiro SUGIYAMARetsu MIURAToshio YAMANO
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1979 Volume 86 Issue 1 Pages 213-223

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Abstract

The reaction processes of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of a new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (kobs values were 20.5 and 16.0s-1) These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermediate is caused by a two-electron transfer process from the reduced pyridine nucle-otide to the enzyme flavin.
On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and with NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semi-quinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR mea-surement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complexed with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.

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© The Japanese Biochemical Society
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