FAD Analogues as Mechanistic and ‘Binding‐Domain’ Probes of Spinach Ferredoxin‐NADP+ Reductase

Abstract

The native flavin, FAD, of spinach ferredoxin, NADP+ reductase, was replaced by a number of FAD analogs 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These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with othe properties of this class, e.g., stabilization of the neutral flavin semiquinone. The chemical reactivity toward methylmethanethiolsulfonate of the 8-mercapto-FAD.cntdot.enzyme clearly shows that the flavin 8-position is exposed to solvent. The lack of reactivity with the 2-thio-FAD.cntdot.enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The 7 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.