The Stability, Reduction Potential and Ligand State of Two Conformations of a C-Type Cytochrome from the Diatom Thalassiosira Pseudonana

Abstract

In this study we investigate the structure and function of a c-type cytochrome from the diatom Thalassiosira pseudonana, Tp34211. The axial iron ligand of a c-type cytochrome is often either a methionine or histidine residue. The axial ligand plays an important role in determining the midpoint potential of the heme cofactor, which in turn determines the possible physiological electron donors and acceptors. Initial experiments suggested that recombinantly expressed Tp34211 forms two alternative conformations that can be differentiated by Soret band changes upon imidazole binding or quenching of the tryptophan fluorescence emission signal. Based on sequence alignments of Tp34211 and known cytochrome c6 proteins, we predict that Tp34211 has an axial histidine ligand (H78), but a neighboring lysine residue (K79) could serve as an alternate ligand. To test the possible role of ligand switching in the two alternative conformations observed for Tp34211, we recombinantly expressed and purified both wild-type Tp34211 and a K79A variant that should force bis-His ligation. Following purification by ion-exchange and size-exclusion chromatogrpahy, guanidine induced unfolding assays were performed on the various protein samples, with extent of unfolding measured via intrinsic tryptophan fluorescence. The ΔG°unfolding of the unquenched conformation of WT Tp34211 and the K79A mutant were determined to be the same within error, suggesting that histidine coordination is favored under normal solution conditions. Midpoint potentials of the K79A variant and the two conformations of the wild-type protein will also be measured to further identify the role of histidine versus lysine ligation in Tp34211.