BACKGROUND: The reactive oxygen-generating NADPH oxidases (Noxes) function in a 
variety of biological roles, and can be broadly classified into those that are 
regulated by subunit interactions and those that are regulated by calcium. The 
prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic 
subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the 
integral membrane protein, p22phox, and this heterodimer binds to the regulatory 
subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering 
superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 
(NOXA1), respective homologs of p47phox and p67phox, together with p22phox and 
Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also 
regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have 
assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs 
from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a 
choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary 
history of these subunits.
RESULTS: Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the 
metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, 
the unicellular organism that is the closest relatives of multicellular animals, 
encodes early prototypes of p22phox, p47phox as well as the earliest known 
Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are 
seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia 
gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. 
Duplication of primordial p47phox and p67phox genes occurred in vertebrates, 
with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of 
characteristic domains of regulatory subunits suggests a novel view of the 
evolution of Nox: in fish, p40phox participated in regulating both Nox1 and 
Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became 
independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an 
autoinhibitory region that is characteristic of mammalian p47phox, and this was 
subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence 
comparisons identified both putative key residues conserved in characteristic 
domains and previously unidentified conserved regions. Also, candidate 
organizer/activator proteins in fungi and amoeba are identified and hypothetical 
activation models are suggested.
CONCLUSION: This is the first report to provide the comprehensive view of the 
molecular evolution of regulatory subunits for Nox enzymes. This approach 
provides clues for understanding the evolution of biochemical and physiological 
functions for regulatory-subunit-dependent Nox enzymes.