Cytochrome c at model membrane surfaces: Exploration via second harmonic generation-circular dichroism and surface-enhanced resonance Raman spectroscopy

The novel nonlinear optical method of second harmonic generation-circular d ichroism (SHG-CD) has been used to follow the adsorption and redox properti es of a peripheral membrane protein, horse heart cytochrome c, adsorbed at several model membrane surfaces. The SHG-CD response is shown to be affecte d by the oxidation state of the heme within surface-adsorbed cytochrome c, as is consistent with the sensitivity of SHG to the chirality of the heme. SHG-CD measurements show that adsorbed cytochrome c is reducible by ascorba te at alkanethiol surfaces, but not at phospholipid/alkanethiol hybrid bila yer membranes (HBMs). The adsorption of cytochrome c at the model membrane surfaces was verified by surface plasmon resonance. Surface-enhanced resona nce Raman measurements show that cytochrome c adsorbed on a hybrid bilayer membrane retains the Fe-heme conformation associated with solution-phase cy tochrome c and is reducible by applying potential to the supporting electro de. The inability of ascorbic acid to reduce cytochrome c associated with t he HEM is attributed not to a change in its redox potential, but rather to the nature of the interaction of cytochrome c with the HBM.