FIGURE 9.

Nitric-oxide synthase activity in a reconstitution assay with the flavoproteins FLDR and YkuN. NO synthesis by wild-type and mutant proteins W66H and W66F was assessed by measuring total nitrite production in a reconstitution assay (FLDR/YkuN/NOS, 2.5:10:1.0), as described previously (20). The values correspond to reactions carried out in the presence of 400 μm NOHA and 100 μm H₄B or H₄T and 1 mm NAPDH. Values are expressed as mean ± S.D. of triplicate experiments for each condition. Both W66H and W66F displayed a lower yield of NO synthesis compared with wild-type BsNOS, either with H₄B or H₄T.