DOI: 10.1002/chem.201202398 Striking Improvement in Peroxidase Activity of Cytochrome c by Modulating Hydrophobicity of Surface-Functionalized Gold Nanoparticles within Cationic Reverse Micelles Subhabrata Maiti, Krishnendu Das, Sounak Dutta, and Prasanta Kumar Das* [a] Introduction Identification of the liaison between conformational transi- tions of cytochrome c (cyt c; bound at the inner membrane of mitochondria) and its peroxidase activity is an important research area towards understanding different cellular proc- esses, from preventing cell damage by scavenging reactive oxygen species (superoxide anions, hydrogen peroxide, hy- droxide radicals) to apoptotic initiation pathways. [1–4] The presence of lipids activates the peroxidase activity of cyt c at the membrane of the cell/organelle (for example, in the mi- tochondrial membrane, peroxidation of cardiolipin by cyt c initiates the apoptotic program) due to the loss of its native globular structure. [5, 6] Unfolding of the tertiary structure of cyt c facilitates the accessibility of the peroxide molecules into the hexacoordinated heme-iron catalytic site. Peroxide replaces the methionine (Met80) ligand from the heme site that increases the peroxidative activity of cyt c, which obvi- ously has potential importance in different organic reactions like the oxidative polymerization of phenol, aniline, and so on. [6b,d] Improvement in the peroxidase activity of positively charged cytochrome c at physiological pH value due to anionic-lipid-induced conformational change has been shown in different biological model systems. [6] Cyt c under- goes a conformational change in its tertiary structure due to involvement of both hydrophobic and electrostatic interac- tions between the protein and lipid membrane, but it keeps its a-helical conformation intact. [6] To this end, membrane- mimetic systems like reverse micelles of negatively charged surfactants (dioctyl sodium sulfosuccinate, AOT) have been reported to assist in conformational perturbation of cyt c, with the result of higher catalytic activity than that in aque- ous medium. [6b,d] In compartmentalized self-assemblies, such as reverse mi- celles or water-in-oil (w/o) microemulsions, cyt c prefers to be solubilized at the interfacial region because of its propen- sity to be bound at the membrane. [7, 8] This interfacial area is a more congested region than the water pool of reverse mi- celles owing to the presence of surfactant head-groups, Abstract: This work demonstrates a re- markable enhancement in the peroxi- dase activity of mitochondrial mem- brane protein cytochrome c (cyt c) by perturbing its tertiary structure in the presence of surface-functionalised gold nanoparticles (GNPs) within cetyltri- methylammonium bromide (CTAB) re- verse micelles. The loss in the tertiary structure of cyt c exposes its heme moiety (which is buried inside in the native globular form), which provides greater substrate (pyrogallol and H 2 O 2 ) accessibility to the reactive heme resi- due. The surfactant shell of the CTAB reverse micelle in the presence of co- surfactant (n-hexanol) exerted higher crowding effects on the interfacially bound cyt c than similar anionic sys- tems. The congested interface led to protein unfolding, which resulted in a 56-fold higher peroxidase activity of cyt c than that in water. Further pertur- bation in the proteins structure was achieved by doping amphiphile-capped GNPs with varying hydrophobicities in the water pool of the reverse micelles. The hydrophobic moiety on the surface of the GNPs was directed towards the interfacial region, which induced major steric strain at the interface. Conse- quently, interaction of the protein with the hydrophobic domain of the amphi- phile further disrupted its tertiary structure, which led to better opening up of the heme residue and, thereby, superior activity of the cyt c. The cyt c activity in the reverse micelles propor- tionately enhanced with an increase in the hydrophobicity of the GNP-cap- ping amphiphiles. A rigid cholesterol moiety as the hydrophobic end group of the GNP strikingly improved the cyt c activity by up to 200-fold relative to that found in aqueous buffer. Fluo- rescence studies with both a trypto- phan residue (Trp59) of the native pro- tein and the sodium salt of fluorescein delineated the crucial role of the hy- drophobicity of the GNP-capping am- phiphiles in improving the peroxidase activity of cyt c by unfolding its tertiary structure within the reverse micelles. Keywords: cytochrome c · gold · hydrophobicity · nanoparticles · peroxidases · reverse micelles [a] S. Maiti, K. Das, S. Dutta, Prof. P.K. Das Department of Biological Chemistry Indian Association for the Cultivation of Science Jadavpur, Kolkata 700032 (India) Fax: (+ 91) 33-24732805 E-mail: bcpkd@iacs.res.in Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201202398. Chem. Eur. J. 2012, 00,0–0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& FULL PAPER