Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation

Abstract

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287–3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities. Those F65 mutants showing diminished stability (i.e., F65A and F65I) have previously been shown (Hurley JK, et al., 1993b, Biochemistry 52:9346–9354) to be severely impaired kinetically in their electron transfer (ET) reaction with ferredoxin:NADP ⁺ reductase (FNR), a physiological reaction partner of Vfd. Mutants F65W and F65Y, which, as noted, are like wt in stability, also functioned like wt in the ET reaction with FNR (Hurley JK, et al., 1993a, J Am Chem Soc 115:11698–11701). Possible reasons for this correlation between ET properties of the F65 mutants and their conformational stabilities are discussed.