Electron transfer and ligand binding to cytochrome c' immobilized on self-assembled monolayers

We have successfully immobilized Allochromatium vinosum cytochrome c' on carboxylic acid-terminated thiol monolayers on gold and have investigated its electron-transfer and ligand binding properties. Immobilization could only be achieved for pH's ranging from 3.5 to 5.5, reflecting the fact that the protein is only sufficiently pos. charged below pH 5.5 (pI = 4.9). Upon immobilization, the protein retains a near-native conformation, as is suggested by the obsd. potential of 85 mV vs SHE for the heme FeIII/FeII transition, which is close to the value of 60 mV reported in soln. The electron-transfer rate to the immobilized protein depends on the length of the thiol spacer, displaying distance-dependent electron tunneling for long thiols and distance-independent protein reorganization for short thiols. The unique CO-induced dimer-to-monomer transition obsd. for cytochrome c' in soln. also seems to occur for immobilized cytochrome c'. Upon satn. with CO, a new anodic peak corresponding to the oxidn. of an FeII-CO adduct is obsd. CO binding is accompanied by a significant decrease in protein coverage, which could be due to weaker electrostatic interactions between the self-assembled monolayer and cytochrome c' in its monomeric form as compared to those in its dimeric form. The obsd. CO binding rate of 24 M-1 s-1 is slightly slower than the binding rate in soln. (48 M-1 s-1), which could be due to electrostatic protein-electrode interactions or could be the result of protein crowding on the surface. This study shows that the use of carboxyl acid-terminated thiol monolayers as a protein friendly method to immobilize redox proteins on gold electrodes is not restricted to cytochrome c, but can also be used for other proteins such as cytochrome c'.