Dh. Murgida et P. Hildebrandt, Heterogeneous electron transfer of cytochrome c on coated silver electrodes. Electric field effects on structure and redox potential, J PHYS CH B, 105(8), 2001, pp. 1578-1586
Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolaye
rs (SAM) of omega -carboxylalkanethiols that were covalently attached to Ag
electrodes. Employing surface-enhanced resonance Raman (SERR) spectroscopy
, the redox equilibria and the structural changes of the adsorbed Cyt-e wer
e analyzed quantitatively for SAMs of different chain lengths ranging from
2-mercaptoacetic acid (C-2-SAM) to 16-mercaptohexadecanoic acid (C-16-SAM).
In the presence of Cyt-c in the bulk solution, the SERR spectra of the ads
orbed Cyt-c display the characteristic vibrational band pattern of the nati
ve protein conformation denoted as state B1. The enhancement of the SERR si
gnals decreases with increasing chain length, but even at distances as larg
e as 24 Angstrom (C-16-SAM), SERR spectra of high quality could be obtained
. Conversely, no SERR signals could be detected for SAMs including hydroxyl
instead of carboxylate headgroups, implying that Cyt-c is adsorbed via ele
ctrostatic interactions. On the basis of potential-dependent SERR experimen
ts, the redox equilibria of the adsorbed Cyt-c (B1) were analyzed, revealin
g ideal Nernstian behavior (n congruent to 1). However, the redox potential
s exhibit negative shifts compared to that of Cyt-c in solution, which incr
ease with the chain length of the SAMs. In the absence of excess Cyt-c in s
olution (i.e., 0.2 muM), a new conformational state B2 of the adsorbed Cyt-
c is observed. This state B2, which differs from the native state B1 by the
heme pocket structure, includes three substates of different spin and coor
dination configurations. The distribution among these substates as well as
the total contribution of state B2 varies with the chain length of the SAM
such that the latter decreases from 73% at C-2-SAM to 0% at C-11- and C-16-
SAMs. These results imply that the formation of B2 is induced by the electr
ic field at the binding site, generated by the potential drop across the el
ectrode/SAM interface. When an electrostatic model for the interfacial pote
ntial distribution for the electrode/ SAM/protein device is employed, both
the redox potential shifts and the electric-field-induced structural change
s can be consistently explained. The impact of these findings for the proce
sses of Cyt-c at biological interfaces is discussed.