1068-1620/02/2805- $27.00 © 2002 MAIK “Nauka /Interperiodica” 0371 Russian Journal of Bioorganic Chemistry, Vol. 28, No. 5, 2002, pp. 371–384. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 5, 2002, pp. 411–425. Original Russian Text Copyright © 2002 by Zaitseva, Ovchinnikova, Vodovozova, Molotkovsky, Polyakov, Titov, Esipov, Grinkevich. 1 INTRODUCTION Mitochondrial H + -ATPase (ATP synthase, F 1 F 0 - Äíêase, or F 1 F 0 -complex; EC 3.6.1.34) catalyzes the synthesis and hydrolysis of ATP that are conjugated with proton transfer. 2 This membrane-bound complex can be divided into two structural and functional sub- complexes: the peripheral (F 1 or F 1 -ATPase) that car- ries the sites of synthesis and hydrolysis of ATP and the membrane subcomplex (F 0 or the membrane domain F 0 ) that transports protons through the membrane. Both subcomplexes have a complex polypeptide composi- tion [1]. The mitochondrial F 1 -ATPase has been studied most extensively, and its three-dimensional structure was determined by X-ray diffraction analysis [2]. The 1 Please send correspondence to phone: +7 (095) 939-1268; e-mail: grin@soil.msu.ru. 2 Abbreviations: BSA, bovine serum albumin; [ 14 C]DCADP and [ 125 I]IDCOUP, 1-acyl-2-[12-[diazocyclopentadiene-2-carbonyl- amino)-[12- 14 C]dodecanoyl] and 1-acyl-2-[11-([ 125 I]diazo- iodocyclopentadiene-2-carbonyloxy)undecanoyl]-derivatives of sn-glycero-3-phosphocholine, where acyl is a mixture of the resi- dues of palmitic (70%) and stearic (30%) acids; DHB, 2,5-dihy- droxybenzoic acid; MALDI, matrix-assisted laser desorption/ion- ization; OSCP, the subunit of the mitochondrial H + -ATPase that provide it with oligomycin sensitivity; PMSF, phenylmethane- sulfonyl fluoride; PVDF-membrane, poly(vinylidene difluoride) membrane; TEMED, N,N,N',N'-tetramethylethylenediamine; THAP, 2,4,6-trihydroxyacetophenone; [ 125 I]TID, 3-(trifluorome- thyl)-3-(m-[ 125 I]iodophenyl)diazirine; and tricine, N- [tris(hydroxymethyl)methyl]glycin. structure of the membrane domain F 0 is less studied. It is currently believed that the F 0 subcomplex of bovine heart mitochondria consists of at least ten subunits (a, b, c, d, e, f, g, OSCP, F 6 , and A6L), the stoichiometry and topography of which are not understood com- pletely. In this study, we used various lipophilic photoreac- tive labels to identify the F 0 subunits that contact the lipids of the mitochondrial inner membrane. RESULTS AND DISCUSSION At the first step of our study, we used [ 14 C]DCADP as a photoactivatable hydrophobic probe. The acyl group of this probe comprises the residues of palmitic (70%) and stearic (30%) acids. We synthesized this probe as described in [3, 4]. It is known that, upon UV- irradiation of photoreactive lipids of this type, their di- azocyclopentadiene group generates a carbene, which exhibits a much higher reactivity than the aromatic nitrenes formed upon photoactivation of conventional phenylazide labels and can therefore be incorporated in a high yield into inactive C–H bonds [5]. Due to a high reactivity, this carbene has a low selectivity toward neighboring molecules, which enhances the successful use of such lipid probes for the nearest neighbor analy- sis. Mitochondrial H + -ATPase: Identification of Subunits of the F 0 Subcomplex that Contact Membrane Lipids L. G. Zaitseva*, T. V. Ovchinnikova*, E. L. Vodovozova*, Yul. G. Molotkovsky*, N. B. Polyakov**, M. I. Titov*, S. E. Esipov*, and V. A. Grinkevich** 1 *Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 10/16, GSP Moscow, 117997 Russia **Chair of Bioorganic Chemistry, Biological Faculty, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia Received September 10, 2001; in final form, December 4, 2001 Abstract—The subunits of the F 0 membrane sector of bovine heart mitochondrial H + -ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteolipo- somes that contained active mitochondrial H + -ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di- azocyclopentadiene-2-carbonylamino)-[12- 14 C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11- ([ 125 I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12- (diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits Ò and ‡ of the F 0 membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry. Key words: mitochondrial H + -ATPase, subunits of the F 0 membrane sector, photoreactive lipids, MALDI mass spectrometry of membrane proteins