Open AccessFAD Analogues as Mechanistic and ‘Binding‐Domain’ Probes of Spinach Ferredoxin‐NADP + Reductase
Author(s) -
ZANETTI Giuliana,
MASSEY Vincent,
CURTI Bruno
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07348.x
Subject(s) - flavin group , ferredoxin , ferredoxin—nadp(+) reductase , chemistry , semiquinone , stereochemistry , redox , dehydrogenase , electron acceptor , enzyme , photochemistry , biochemistry , quinone , organic chemistry
The native flavin, FAD, of spinach ferredoxin—NADP + reductase, has been replaced by a number of FAD analogues with modifications of the isoalloxazine ring system. The apoenzyme binds 8‐mercapto‐FAD in its thiolate anion form and 6‐hydroxy‐FAD in its neutral form. These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with other properties of this class, e. g. stabilization of the neutral flavin semiquinone. The chemical reactivity toward methylmethanethiolsulfonate of the 8‐mercapto‐FAD enzyme clearly shows that the flavin 8‐position is exposed to solvent. On the other hand, the lack of reactivity with the 2‐thio‐FAD enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The seven modified flavins examined all support NADPH‐ferricyanide reductase activity, the catalytic velocity being directly proportional to the redox potential of the flavin. No such linear free energy relationship was found between redox potential and activity with ferredoxin or iodonitrotetrazolium as acceptor.