Cytochrome c Oxidase Inhibition by N-Retinyl-N- retinylidene Ethanolamine, a Compound Suspected to Cause Age-Related Macula Degeneration Hamdy Shaban, Paolo Gazzotti, and Christoph Richter 1 Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Universita ¨ tstr. 16, CH-8092 Zurich, Switzerland Received July 5, 2001, and in revised form July 23, 2001; published online September 12, 2001 The endogenous lipophilic and cationic compound N-retinyl-N-retinylidene ethanolamine (A2E) is sus- pected to cause age-related macula degeneration. It inhibits cytochrome c oxidase, detaches proapoptotic proteins from mitochondria, and induces apoptosis in mammalian retinal pigment epithelial cells (M. Suter, C. E. Reme ´ , C. Grimm, A. Wenzel, M. Ja ¨a ¨ ttela, P. Esser, N. Kociok, M. Leist, and C. Richter, 2000, J. Biol. Chem. 275, 39625–39630). The inhibition of cytochrome c oxi- dase is highly specific for A2E and is observed with the solubilized and reconstituted enzyme. In the dark, in- hibition is overcome by cardiolipin or other acidic phospholipids. With illumination, inhibition is stron- ger, becomes complete with prolonged exposure, and is then no longer abrogated by cardiolipin. Cardio- lipin effectively displaces A2E from cytochrome c oxi- dase, suggesting noncovalent binding of A2E to the enzyme. We conclude that A2E is a potent cytochrome c oxidase-specific inhibitor which interferes with the binding of cytochrome c to cytochrome c oxidase and, in the light, causes persistent modifications of the en- zyme. © 2001 Academic Press Key Words: age-related macula degeneration; A2E; apoptosis; cardiolipin; cytochrome c; cytochrome c oxidase. Age-related macular degeneration (AMD) 2 affects about one-fifth of the population older than 65 years and is one of the main causes of poor vision in the elderly in industrialized nations (1). It is accompanied by photoreceptor damage in the macula. The primary lesion in AMD appears to reside in the retinal pigment epithelium (RPE), possibly resulting from its high rate of degradation. A better understanding of the patho- genesis of AMD is needed to successfully prevent and/or treat this disease. Accumulation of the autofluorescent age pigment li- pofuscin in RPE cell phagolysosomes is a predicament for the development of AMD. Lipofuscin harbors two retinoids, the lipophilic cations N-retinyl-N-retinyli- dene ethanolamine (A2E) and its isoform, iso-A2E, first isolated from eyes of old humans (2, 3). The molecules can be synthesized in vitro from two retinals and one ethanolamine (3), both components of the photorecep- tor outer segment membranes, where 11-cis-retinal serves as the chromophore of the visual pigment rho- dopsin and phosphatidylethanolamine is an abundant membrane phospholipid. We recently showed that A2E induces apoptosis in cultures of various mammalian cell types, including RPE cells (4). A2E-induced apoptosis is preceeded by a decline in mitochondrial activity and accompanied by translocation of cytochrome c and apoptosis-inducing factor into the cytoplasm and nucleus. A2E, but not iso-A2E, targets the function of cytochrome c oxidase, whereas respiratory chain activity upstream of cyto- chrome c is not affected by A2E. cytochrome c oxidase activity can be restored by added cardiolipin (1,3- diphosphatidylglycerol, 1,3-DPG). Here we investigate in more detail the inhibition of cytochrome c oxidase by A2E. Inhibition is highly spe- 1 To whom correspondence should be addressed: Fax: +41-1- 6321121. E-mail: richter@bc.biol.ethz.ch. 2 Abbreviations used: AMD, age-related macular degeneration; A2E, N-retinyl-N-retinylidene ethanolamine, 2-[2,6-dimethyl-8- (2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1- (2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)- 1E,3E,5E-hexatrienyl]-pyridinium; 1,3-DPG, 1,3-diphosphatidyl- glycerol, cardiolipin; IC 50 , concentration necessary to achieve 50% inhibition; RPE, retinal pigment epithelium; TMPD, tetramethyl-p- phenylenediamine. 0003-9861/01 $35.00 111 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 394, No. 1, October 1, pp. 111–116, 2001 doi:10.1006/abbi.2001.2535, available online at http://www.idealibrary.com on