Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the a and the b subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase.Despite harsh treatments with, e.g. detergents and denaturing agents, the a and b subunits remained tightly associated. A monoclonal antibody directed towards the a subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II.Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD 1 by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.Keywords: transhydrogenase; NADP, NAD; membrane protein; proton pump. (Fig. 1). The active forms of the enzymes are dimers (220 kDa), either as a homodimer in the case of the mitochondrial H 1 -TH, as (a,b) 2 in the case of the Escherichia coli H 1 -TH or as (a1,a2,b) 2 in the case of Rhodospirillum rubrum H 1 -TH (1). As shown in Fig. 1, the a subunit (54 kDa) of E. coli H 1 -TH comprises domain I (40 kDa) and four transmembraneous helices, which form one component of dII. The b subunit (49 kDa) is composed of domain III (20 kDa) and nine helices spanning the membrane which constitute the second part of dII. The a1 subunit of R. rubrum corresponds only to domain I and is therefore purified as a soluble homodimer of 81 kDa [3]. The structural properties of domain I (dI) and domain III (dIII), from the three sources mentioned previously, have been characterized by expressing them separately. The determination of their structures has been realized by structural predictions [4±6], NMR [6±9] and X-ray crystallography [10,11]. From these studies, it was found that dI was made of two similar domains (dIa and dIb) each of which comprise a parallel b sheet surrounded by mainly b helices. The structure of domain III also exhibited a classical dinucleotide-binding fold [10,11].