Dissolved organic matter in water of Daugava river

Fluorescence Study of Cytochrome c Binding to Model MembranesV.Trusova *1 , G.Gorbenko 1 , J.Molotkovsky 2 , P.Kinnunen 31 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;3 School of Science and Technology, Aalto University, Espoo, Finlande-mail: valtrusova@yahoo.comCytochrome с (cyt c) is a small highly basic hemoprotein abundant in the intermembranespace of mitochondria and possessing two at first glance unrelated functions. First, cyt с isan essential component in respiration, shuttling electrons from cyt c reductase (bc1complex) to cyt c oxidase. The second function of cyt c relates to its key role in theactivation of the apoptotic cascade, resulting from the release of this protein frommitochondria into the cytosol. It is generally established that control of the binding of cytc to the inner membrane of mitochondria (IMM) is involved in both of the abovefunctions. Despite the diversity of lipids constituting the mitochondrial membrane, centralto the cyt c – membrane interaction in IMM is cardiolipin (CL), which is indispensible forthe retention, stability and physiological functioning of cyt c. In this regard, the nature andspecificity of interactions between cyt c and CL attract particular interest. In the presentstudy fluorescence resonance energy transfer (FRET) between anthrylvinyl-labeledphosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptorhas been employed to explore the protein binding to model membranes, composed ofphosphatidylcholine (PC) and CL. The existence of two types of protein-lipid complexeshas been hypothesized where either deprotonated or partially protonated CL molecules areresponsible for cyt c attachment to bilayer surface. To quantitatively describe cyt cmembrane binding, the adsorption model based on scaled particle and double layertheories has been employed, with potential-dependent association constants being treatedas a function of acidic phospholipid mole fraction, degree of CL protonation, ionicstrength and surface coverage. Multiple arrays of FRET data obtained under conditions ofvarying pH, ionic strength, CL content and protein-to-lipid molar ratio have been analyzedin terms of the model of energy transfer in two-dimensional systems combined with theadsorption model allowing for area exclusion and electrostatic effects. The set ofrecovered model parameters included effective protein charge, intrinsic associationconstants and heme distance from the bilayer midplane for both types of protein-lipidcomplexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close tothat characteristic of IMM) the binding equilibrium dramatically shifted towards cyt cassociation with partially protonated CL species. The estimates of heme distance frombilayer center suggest shallow bilayer location of cyt c at physiological pH, while at pHbelow 6.0 the protein tends to insert into membrane core. These findings support theviewpoint that hydrogen bonding of cyt c to protonated phosphate can be coupled with theformation of electrostatic contacts with deprotonated phosphate. Structural features of cytc do not exclude such a possibility. Cyt c molecule has two hydrophobic channelsconnecting heme crevice with the protein surface. The opening of one of these channels issurrounded by oppositely located Asn 52 and Lys 72 , Lys 73 . It has been hypothesized that cytc association with CL involves hydrogen bonding of one protonated phosphate moiety toAsn 52 with simultaneous electrostatic binding of deprotonated phosphate to lysine residuesand accommodation of one acyl chain within the hydrophobic channel.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 32 -