Circular Dichroism Studies of Glutathione Reductase

The circular dichroic spectrum of glutathione reductase was studied. The far-ultraviolet region of the spectrum indicated a low content of helix and β structures, as calculated on tire basis of commonly used spectral parameters of secondary structure. The fractions of amino acid residues present in helix and β structures, respectively, constitute approximately 30% each of the total number in crystals of the enzyme from human erythrocytes [G. E. Schulz, R. H. Schirmer, W. Sachsenheimer, and E. F. Pai (1978) Nature (Lond.) 273, 120–124]. This discrepancy between the estimates may originate from the presence of supersecondary structures in the enzyme, which invalidate the use of parameters defined for circular dichroic spectra of simple secondary structures. Nevertheless, the similarities between the spectra of glutathione reductase from calf liver, porcine erythrocytes, and human erythrocytes strongly suggest that the secondary and supersecondary structures of the three enzymes are very similar. Furthermore, the far-ultraviolet circular dichroic spectrum of the apoenzyme is not significantly different from that of the holoenzyme, indicating lack of major structural differences between the two forms of the protein. The circular dichroism of the near-ultraviolet region indicates changes in the interaction of FAD with the protein matrix as well as local conformational changes involving aromatic side-chains of amino acid residues during redox changes of the holoenzyme. Differences between near-ultraviolet spectra of apoenzyme and holoenzyme likewise indicate reversible local conformational changes upon binding and release of the prosthetic group FAD. The reconstituted holoenzyme had a spectrum indistinguishable from the native enzyme. The 300–600-nm region of the spectrum could be resolved into six Gaussian functions corresponding to the vibronic bands identified in other flavin-dependent dehydrogenases. The rotational strength of the Cotton effects in the 300–600-nm region increased and the positions of the maxima in the circular dichroism spectrum shifted upon reduction of the enzyme The increase in the 400–510-nm region indicates a stronger asymmetric interaction between the isoalloxazine ring of FAD and the protein than that observed in the oxidized form of the enzyme. The enhancement may be effected by the dithiol, which is formed upon reduction of the enzyme.