Biochimie (1990) 72, 25-32 (~) Soci6t6 de Chimie biologique/Elsevier, Paris 25 Induction by different thioredoxhls of ATPase activity in coupling factor 1 from spinach chloroplasts JM Galmiche 1., G Girault1, G Berger 1, JP Jacquot2, M Miginiac-Maslow2, E Wollman3 tService de Biophysique, CEN Saclay, 91191 Gif-sur-Yvette; 2Laboratoire de Physiologie Vdgdtale Moldculaire et Laboratoire de Photosynthdse et M~tabolisme, UA 1128 CNRS, Universitd de Paris-Sud, 91405 Orsay Cedex; 3Laboratoire d'Immunologie, UA 1156 CNRS, INSERM, Institut Gustave Roussy, 94805 Villejuif Cedex, France (Received 19 October 1989; accepted 9 November 1989) Summary - - ATPase activity of the coupling factor 1, CFl, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF~ must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by hea,t~ng CF~ or removing the inhibitory intrinsic epsilon subunit from CF~. Efficiencyand primary structure of the different thioredoxins were compared. The progressive addition of KCI during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coil thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CFt and are involved in the dual effect of KCi. The variations in the screening effect of the negative charges of the CF~ surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coil or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coil thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coil or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concen- tration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CFs. chloroplasts / coupling factor 1 / ATPase activation spinach / £seheriehia eoU / human lymphocytes / thioredoxins Introduction The chloroplast ATP synthase which catalyses ATP synthesis during photophosphorylation is a proton ATPase of the F1Fo type. The extramembraneous solu- ble part of this ATP synthase or coupling factor 1, CF1, is a latent ATPase which is activated by physical (heat), chemical (reducing thiols, alcohols, detergents) or enzymatic (trypsin) treatment [1]. ATP synthase in vivo is also submitted to regulatory processes. Two activated states of the enzyme are described, one oxidized and the other reduced [2]. Two important findings have helped to elucidate the activation process. First, it was shown that reduced spinach thio- redoxin further enhanced the ATPase activity of the heat-treated CF~ [3]. It was proposed that thioredoxin played a central role in the modulation of CF~ ATPase activity in intact chloroplasts [4]. Secondly, the reduc- tion of a disulphide bond in the gamma subunit of the CF1 was demonstrated to induce its ATPase activity [5-7]. This disulphide bond was shown to be accessible to reducing agents only in the oxidized activated state of CF1 where the intrinsic inhibitory subunit epsilon was lost or had modified connection with the other subunits of the enzyme. Such a state was obtained by heating the CF 1. Moreover, epsilon subunit deprived CFl behaved as an oxidized activated ATPase and after reduction of the disulphide bond of its gamma subunit exhibited enhanced rate of ATP hydrolysis. We used the preparation by HPLC of epsilon deprived CF 1 [8] to study the activation processes by reduction of the disulphide bond by reduced thioredoxins from higher plants, green algae, bacteria and human lymphocytes. Comparisons between their activities and *Correspondence and reprints Abbreviations: AA, amino acid; CFI, coupling factor 1; D, aspartate; DTI', dithiothreitol; E, glutamate; EDTA, ethylenediaminetetraacetic acid; K, lysine; R, arginine; Tx Ch, thioredoxin from Chlamydomonas chloroplasts; 'Ix E, thioredoxin from Escherichia coli; 'Ix H, thioredoxin from human iymphocytes; Tx m, thioredoxin m from spinach chloroplasts