Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential i
ron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (
Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and ala
nine (Y12A). Variants Y12A and Y12I were expressed to reasonable level
s in cells grown at lower temperatures, but decomposed during purifica
tion. Variants Y12F, Y12H, and Y12W were substantially destabilized wi
th respect to the recombinant wild-type HiPIP (rcWT) as determined by
differential scanning calorimetry over a pH range of 7.0-11.0. Charact
erization of the Y12F variant by NMR indicates that the principal stru
ctural differences between this variant and the rcWT HiPIP result from
the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with
Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss o
f the latter interaction is propagated through the Lys-59/ Val-58 pept
ide bond, thereby perturbing Gly-46. The Delta Delta G(D)(app) of Y12F
of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is
entirely consistent with the contribution of these two hydrogen bonds
to the stability of the latter. CD measurements show that Tyr-12 influ
ences several electronic transitions within the cluster. The midpoint
reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and
22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), res
pectively, higher than that of rcWT HiPIP. The current results indicat
e that, although conserved Tyr-12 modulates the properties of the clus
ter, its principle function is to stabilize the HiPIP through hydrogen
bonds involving its hydroxyl group and electrostatic interactions inv
olving its aromatic ring.