Protonation of the Cysteine Axial Ligand Investigated in His/Cys c-Type Cytochrome by UV-Vis and Mid- and Far-IR Spectroscopy.

His/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Toward a better understanding of Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and mid- to far-FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C = -375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys ν(SH), His ν(C5N1), heme δ(CmH)) and far-IR (ν(Fe(II)-His), ν(His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, is stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination.

Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum.

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands beta 5 and beta 6, loop L5 and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.