Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin.

Putidaredoxin (Pdx), a Cys4Fe2S2 ferredoxin from Pseudomonas putida, exhibits redox-dependent binding to its physiological redox partner, cytochrome P450(cam) (CYP101), with the reduced form of Pdx (Pdx(r)) binding with greater affinity to oxidized camphor-bound CYP101 than the oxidized form, Pdx(o). It has been previously shown that Pdx(o) is more dynamic than Pdx(r) on all accessible time scales, and it has been proposed that Pdx(r) samples only a fraction of the conformational substates populated by Pdx(o) on a time average. It is postulated that the ensemble subset populated by Pdx(r) is the same subset that binds CYP101, providing a mechanism for coupling the Pdx oxidation state to binding affinity for CYP101. Evidence from a variety of sources, including redox-dependent shifts of 15N and 13C resonances, indicates that the metal cluster binding loop of Pdx is the primary determinant of redox-dependent conformational selection. Patterns of paramagnetic effects suggest that the metal cluster binding loop contracts around the metal cluster upon reduction, possibly due to the strengthening of hydrogen bonds between the sulfur atoms of the metal cluster and the surrounding polypeptide NH and OH groups. Effects of this perturbation are then transmitted mechanically to other affected regions of the protein. A specific mutation has been introduced into the metal binding loop of Pdx, G40N, that slows conformational exchange sufficiently that the ensemble of conformational substates in Pdx(o) are directly observable as severe broadenings or splittings in affected NMR resonances. Many of the residues most affected by the mutation also show significant exchange contributions to 15N T(2) relaxation in wild-type Pdx(o). As predicted, G40N Pdx(r) shows a collapse of many of these multiplets and broadened lines to form much sharper resonances that are essentially identical to those observed in wild-type Pdx(r), indicating that Pdx(r) occupies fewer conformational substates than does Pdx(o). This is the first direct observation of such redox-dependent ensembles at slow exchange on the chemical shift time scale. These results confirm that conformational selection within the Fe2S2 cluster binding loop is the primary source of redox-dependent changes in protein dynamics in Pdx.