THE ROLE OF A CONSERVED TYROSINE RESIDUE IN HIGH-POTENTIAL IRON-SULFUR PROTEINS

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.