Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima

The kinetics of the irreversible unfolding of glutathione reductase (NAD[P] H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima wa s studied at pH 7.0 and room temperature. Denaturation was induced by guani dinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtai ned, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast ear ly event in the denaturation pathway was the dissociation of tetrameric pro tein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the un folding pathway the latter was dissociated into denatured monomers. Fluores cence measurements revealed that denaturation of S. maxima glutathione redu ctase is a slow process. Release of the prostethic group FAD was previous t o the unfolding of the enzyme. No aggregated species were detected in the u nfolding pathway, dismissing the aggregation of denatured polypeptide chain s as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the d enaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ m ol(-1) was obtained for the activation free energy of unfolding in the abse nce of denaturant. No evidence for the native monomer in the unfolding path way was obtained which suggests that the dimeric nature of glutathione redu ctase is essential for the maintenance of the native subunit conformation. (C) 2001 Academic Press.