Reverse gyrase is a DNA topoisomerase that catalyzes positive DNA supercoiling in an ATP-dependent reaction. The enzyme consists of a helicase and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. For positive supercoiling of DNA, the two domains need to cooperate, presumably through conformational changes. Here, we probed conformational changes of Thermotoga maritima reverse gyrase on DNA and nucleotide binding using hydrogen deuterium exchange (HDX). While ADPNP binding does not induce major conformational changes, binding of DNA leads to increased exposure of a region at the helicase-topoisomerase interface, encompassing R501 and E705, the side chains of which engage in electrostatic interactions. Removing the charges in an R501/E705A variant leads to a moderate increase in supercoiling activity, suggesting a slightly inhibitory role of this interactions. A R501/E705C variant, crosslinked to fix the two side chains in a disulphide bond, on the other hand, is inactive. Collectively these data show that rearrangements at the helicase and topoisomerase interface are important for the functional cooperation of the domains and for the enzymatic activity of reverse gyrase.