Affiliation: | (1) School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA;(2) College of Pharmacy, Chungbuk National University, 361-763 Cheongju , Korea;(3) Department of Chemistry, University of Georgia, Athens, GA 30602-2556, USA;(4) Wilkes Honors College, Jupiter, FL 33458, USA;(5) Department of Chemistry, Georgetown University, Washington, DC 20057-1227, USA |
Abstract: | Rubredoxin is a small iron-sulfur (FeS4) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the S of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between S of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent. |