Redox Properties of CytochromecAdsorbed on Self-Assembled Monolayers: A Probe for Protein Conformation and Orientation

Abstract

The redox behavior of cytochrome c (cyt c) adsorbed to gold electrodes modified with self-assembled monolayers (SAMs) depends on the SAM. This paper examines SAMs generated from alkanethiols terminating in trimethylammonium (1), sulfonate (2), methyl (3), amine (4), and carboxylic acid (5) groups and from an aromatic thiol (6). The redox potentials of cyt c adsorbed on SAMs of 1 and 5 are relatively close to the formal potential of native cyt c measured in solution. The redox potentials of cyt c adsorbed on SAMs of 3, 4, and 6 are significantly shifted from the formal potential, and a reduction peak at about 0.5 V more negative than the formal potential (that is, a value corresponding to a more difficult reduction) was observed in all three cases. These observations suggest that cyt c changes its conformation significantly on adsorption on these surfaces. No redox peaks were observed for cyt c adsorbed on SAMs of 2, although surface plasmon resonance (SPR) studies indicate that the SAMs of 2 irreversibly adsorbed approximately a double layer of cyt c. Mixed SAMs were also studied. Most interestingly, cyt c adsorbed on mixed SAMs formed from the combinations of 1 and 2 exhibited significantly slower electron transfer (0.3-1.2 s -1 ) than cyt c adsorbed on a homogeneous SAM of 1 (45 s -1 ). These observations suggest changes in protein orientation due to the presence of the sulfonate groups at the interface. This study suggests that electrochemical measurement can be a useful probe for the conformation and orientation of protein adsorbed on surfaces.

5-(4-pyridinyl)-1,3,4-oxadiazole-2-thiol on gold: SAM Formation and electroactivity

“…31 Also, the observation of such potential suggests that there is no change in the protein conformation in consequence of the SAM-Cyt c molecular recognition process. 32,33 Based on the Nicholson's method, 34 the dependence of the difference between the anodic and cathodic peak potentials ( E p ) on the scan rate allows one to estimate the kinetic parameter, . Based on equation 3 with f = F/RT, the heterogeneous electron transfer rate constant, k 0 , was determined from the slope of the curve presented in the plot of 2 vs. the reciprocal of the scan rate, v -1 (Figure 3c).…”

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

“…These methods include immobilizing the enzyme in a polyion film (Masuda and Fukuda, 1995), entrapping the enzyme in a sol-gel film (Masuda et al, 2000), incorporating the enzyme into conducting polymer films (Funk et al, 2005), modifying the enzyme with an electrical relay (Hahm and Lieber, 2003), functionalizing the electrode with a biomembrane-like surfactant (Ohldag et al, 2006), and supporting the enzyme in a clay nanoparticle modified electrode (Shumyantseva et al, 2004). However, because these methods do not control enzyme aggregation on the electrode surface, the efficiency of electron transfer to the active site of the enzyme may be reduced (Habermüller et al, 2000;Chen et al, 2002), potentially through altered enzyme conformation.…”

“…For example, alkane-thiol or other thiol-terminated chains covalently bonded to a noble-metal surface and functionalized at the terminus with a group that binds to a specific site of the protein, which have been used to control enzyme binding orientation (Collinson and Bowden, 1992;Pardo-Yissar et al, 2000;Chen et al, 2002). CYP2E1 has been bonded through thiols, both directly or via a cysteine-maleimide linker, to a gold surface although enzyme orientation remained ambiguous (Fantuzzi et al, 2004).…”

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

“…In both the photosynthetic and respiratory chains, the transport of electrons between the enzymes embedded in the membrane is accomplished either by molecules freely diffusing in the membrane, the quinones, or water soluble electron carrying proteins, such as cytochrome (cyt) c. This small monoheme protein (12.5 kDa) [4][5][6], which transfers electrons between cyt bc1 complex and cyt c oxidase in the mitochondrial respiratory chain, has long served as a model system for mechanistic studies of protein ET processes. Important insight into the distance dependence and reorganization energy of the ET process has been gained from protein film voltammetry studies of cyt c immobilized on electrodes modified with various functional groups including alkyl [7], pyridyl [8,9], amino [10,11], carboxyl [12][13][14], L-cysteinyl [15] and hydroxyl [16]. In particular, studies at metallic surfaces modified with -carboxyl alkanethiols by a combined approach of electrochemistry, surface-enhanced and time-resolved spectroscopic techniques have provided a full image of the electron transfer dynamics of electrostatically immobilized cyt c and have highlighted the important role of electric field effects in the ET properties [17][18][19][20][21].…”