Binding of nicotinamide adenine dinucleotide phosphate to the tetratricopeptide repeat domains at the N-terminus of p67PHOX, a subunit of the leukocyte nicotinamide adenine dinucleotide phosphate oxidase

The nicotinamide adenine dinucleotide phosphate (NADPH) binding site of the NADPH oxidase complex is believed to be located on the beta, subunit of cytochrome b(558). However, our previous studies showed that p67(PHOX) also contains an NADPH binding site that is essential for normal oxidase activity and that p67(PHOX) is able to mediate a slow electron transfer from a reduced pyridine nucleotide to an artificial electron acceptor. Using both affinity labeling and fluorescence quenching, we have obtained further evidence that p67(PHOX) is able to bind NADPH. We have used a number of truncated forms of p67(PHOX), including p67(PHOX)(1-243), p67(PHOX)(1-210), p67(PHOX)(1-199), and p67(PHOX)(244-526) (where the numbers represent the initial and final amino acids in the truncated p67(PHOX)) in order to localize the binding site. We found that NADPH could bind to p67(PHOX)(1-243), p67(PHOX)(1-210), and p67(PHOX)(1-199) but not to p67(PHOX)(244-526). The p67(PHOX)(1-199) fragment consists largely of four tetratricopeptide (TPR) domains. We showed further that Rac2-GTP gamma S and to a lesser extent Rac2-GDP beta S could modulate the binding of NADPH to p67(PHOX).