The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed (35)S-eNOS reductase domain but not the (35)S-eNOS oxygenase domain. Furthermore GST-dyn-2 PRD binds a (35)S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.)