covalent stabilization, thus being the dominant interaction. The similarity of the structures 1-3 and of their interactions indicates that NADPH and FAD are arranged in a nearly ideal fashion for a hydride transfer to N-5 of the isoalloxazine ring in the enzyme.
In the second step, FADH' transfers reduction equivalents to the disulfide bridge Cys58:Cys63 (cf. 5, bottom). In this process, the direct nucleophilic attack of the reduced isoalloxazine ring offers an attractive possibility corresponding to the usual proposal of nucleophilic attack on FAD.[91 In such an attack, the nucleophile forms a covalent bond to C-4a of the isoalloxazine. Crystal structure analysis and calculation supports such a mechanism. Especially interesting is the observed unusual conformation of the disulfide bridge,[31 which, in the given fixed position relative to the isoalloxazine ring, leads to an exact orientation of the antibonding LUMo of the bridge toward C-4a, as is shown by the scaled reproduction of the crystal structure of 5. C-4a of the isoalloxazine ring and the S atoms of the disulfide bridge lie next to each other in a linear fashion, and the energetically nearly degenerate frontier orbitals (HOM0,,,,e-LUM0,,,,8:Cys63) result in a strong covalent interaction between C-4a and the sulfur atom of Cys63. Structure 5 serves to emphasize the favorable orientation of the orbitals for back-side attack of C-4a on the disulfide bond. The mechanism can be described as a nucleophilic cleavage of the SS bridge with formation of a covalent bond between C-4a of the isoalloxazine and the S atom of Cys63. Transfer of the proton from N-5 to the S atom of C y ~5 8 ~ and fragmentation by intramolecular nucleophilic substitution to give Cys63" should follow. It is interesting to note that the MNDO PM3 calculation for the interaction of FAD with Cys63@, the relative position of which is known from X-ray data, gives a stabilization of -2.6 kcal mol-' and shows no repulsion, although this is usually observed between molecules with closed valence shells. The calculation apparently confirms the spectroscopically established formation of a CT complex['. 21 between FAD and Cys63'.
The MNDO PM3 calculations and the perturbation analysis of the structure of the active site of glutathione reductase show that, in the enzyme, the reaction partners NADPH and FAD, as well as FADH' and Cys58:Cys63, are positioned in such a way that they are localized near the transition state along the reaction coordinates of the hydride transfer and the nucleophilic cleavage of the disulfide bridge, respectively. This positioning eliminates the decrease in entropy required for this reaction and is in agreement with proposals concerning enzyme-catalyzed reactions in general. Furthermore, other mechanisms, such as one-electron transfers, become improbable. Further investigations, which also consider the reduction of glutathione and include some important amino acids in the vicinity of the active center, are in progress.