MUTATIONS OF SURFACE RESIDUES IN ANABAENA VEGETATIVE AND HETEROCYST FERREDOXIN THAT AFFECT THERMODYNAMIC STABILITY AS DETERMINED BY GUANIDINE-HYDROCHLORIDE DENATURATION
Jk. Hurley et al., MUTATIONS OF SURFACE RESIDUES IN ANABAENA VEGETATIVE AND HETEROCYST FERREDOXIN THAT AFFECT THERMODYNAMIC STABILITY AS DETERMINED BY GUANIDINE-HYDROCHLORIDE DENATURATION, Protein science, 4(1), 1995, pp. 58-64
The stability properties of oxidized wild-type (wt) and site-directed
mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) f
erredoxins from Anabaena 7120 have been characterized by guanidine hyd
rochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E
95K, 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 i
n 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 r
ates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemist
ry 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 crys
tal structures of wt Anabaena Vfd and Hfd, the decreased stabilities o
f F65A and F65I can be ascribed to increased solvent exposure of inter
ior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the
decreased stabilities may result from disruption of a hydrogen bond be
tween 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) h
ave previously been shown (Hurley JK, et al., 1993b, Biochemistry 32:9
346-9354) to be severely impaired kinetically in their electron transf
er (ET) reaction with ferredoxin:NADP(+) reductase (FNR), a physiologi
cal reaction partner of Vfd. Mutants F65W and F65Y, which, as noted, a
re like wt in stability, also functioned like wt in the ET reaction wi
th FNR (Hurley JK, et al., 1993a, J Am Chem Soc 115:11698-11701). Poss
ible reasons for this correlation between ET properties of the F65 mut
ants and their conformational stabilities are discussed.