Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like 
other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding 
domain at its N-terminus, a transmembrane heme-containing region, and a 
C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 
require regulatory subunits, including p22phox, Nox5 activity does not depend on 
any subunits. We found that inactive point mutants and truncated forms of Nox5 
(including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, 
consistent with formation of a dominant negative complex. Oligomerization of 
full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, 
differentially tagged forms of Nox5 and occurred in a manner independent of 
calcium ion. Several approaches were used to show that the DH domain mediates 
oligomerization: Nox5 could be isolated as a multimer when the calcium-binding 
domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion 
of the DH domain eliminated oligomerization. Further, a chimera containing the 
transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase 
(CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex 
with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation 
inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in 
human aortic smooth muscle cells indicated molecular masses of ∼350 and ∼300 
kDa, respectively, consistent with a tetramer being the functionally active 
unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is 
mediated by its dehydrogenase domain. As a result of oligomerization, the short, 
calcium-independent splice form, Nox5S, may function as an endogenous inhibitor 
of calcium-stimulated ROS generation by full-length Nox5.