To understand the regulatory function of the gamma and F subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric 17,171 complex of thermophilic bacteria. When a part of the chloroplast F, gamma subunit was introduced into the bacterial F0F1 complex, the inverted membrane vesicles with this chimeric F0F1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal a-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of F0F1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of F0F1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial F0F1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity. (C) 2004 Elsevier Inc. All rights reserved.