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Electron-transferring flavoprotein from pig kidney: flavin analogue studies.

Abstract：

:Apo-electron-transferring flavoprotein from pig kidney (apo-ETF) has been prepared by an acid ammonium sulfate procedure and reconstituted with FAD analogues to probe the flavin binding site. The 8-position of the bound flavin is accessible to solvent as judged by the reaction of 8-Cl-FAD-ETF with sodium sulfide and thiophenol. A series of 8-alkylmercapto-FAD analogues containing increasingly bulky substituents bind tightly to apo-ETF and can be reduced to the dihydroflavin level by octanoyl-CoA in the presence of catalytic levels of the medium-chain acyl-CoA dehydrogenase. Bulky substituents severely slow the rate of these interflavin electron-transfer reactions. In the case of the 8-cyclohexylmercapto derivative, this decrease reflects a sizable increase in the Km for ETF (approximately 14-fold) with only a 20% decrease in Vmax. Reduction of all of these 8-substituted derivatives involves the accumulation of ETF anion radical intermediates. Dihydro-5-deaza-FAD dehydrogenase, unlike the corresponding 1-deazaflavin substitution, is unable to reduce native ETF despite a strongly favorable redox potential difference. These results, together with data from the native proteins, are consistent with obligatory 1-electron transfer between dehydrogenase and ETF possibly involving the exposed dimethylbenzene edge of ETF. Irradiation of apo-ETF reconstituted with the photoaffinity analogue 8-azidoflavin leads to approximately 10% covalent incorporation of the flavin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of apo-ETF labeled with tritiated 8-azido-FAD shows preferential labeling of the smaller subunit (88%, Mr 30,000 subunit; 12%, Mr 33,000 subunit).(ABSTRACT TRUNCATED AT 250 WORDS)

journal_name

Biochemistry

journal_title

Biochemistry

authors

Gorelick RJ,Thorpe C

doi

10.1021/bi00370a050

subject

Has Abstract

pub_date

1986-11-04 00:00:00

pages

7092-8

issue

22

eissn

0006-2960

issn

1520-4995

journal_volume

25

pub_type

杂志文章

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