Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase.
Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 10(5)) was about 20 times greater than that of YkuP (2.6 x 10(4)), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.
Wang
Zhi-Qiang
ZQ
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. wangz3@ccf.org
Lawson
Rachel J
RJ
Buddha
Madhavan R
MR
Wei
Chin-Chuan
CC
Crane
Brian R
BR
Munro
Andrew W
AW
Stuehr
Dennis J
DJ
D000818:Animals; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D005418:Flavodoxin / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013045:Species Specificity
D001426:Bacterial Proteins; D005418:Flavodoxin; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase