Synchronous photoinitiation of endothelial NO synthase activity by a nanotrigger targeted at its NADPH site.
We designed a new nanotrigger to synchronize and monitor an enzymatic activity interacting specifically with the conserved NADPH binding site. The nanotrigger (NT) combines a docking moiety targeting the NADPH site and a chromophore moiety responsive to light excitation for efficient electron transfer to the protein. Specific binding of the nanotrigger to the reductase domain of the endothelial nitric oxide synthase (eNOSred) was demonstrated by competition between NADPH and the nanotrigger on the reduction of eNOSred flavin. A micromolar Ki was estimated. We had monitored initiation of eNOSred activity by ultrafast transient spectroscopy. The transient absorption spectrum recorded at 250 ps fits the expected sum of the reduced and oxidized species, independently obtained by other chemical methods, in agreement with a photoinduced electron transfer from the excited nanotrigger to the flavin moiety of eNOSred. The rate of electron transfer from the excited state of the nanotrigger (NT*) to the protein is estimated to be k(ET) = (7 +/- 2) x 10(9) s(-1) using the decay of oxidized eNOSred-bound nanotrigger compared against prereduced eNOSred or glucose 6-P dehydrogenase as controls. This fast electron transfer bypasses the slow hydride transfer to initiate NOS catalysis as shown by ultrafast kinetics using the eNOSred mutated in the regulatory F1160 residue. The selective targeting of the nanotrigger to NADPH sites should allow controlled initiation of the enzymatic activity of numerous proteins containing an NADPH site.
Beaumont
Edward
E
Unité 696, INSERM, Palaiseau France.
Lambry
Jean-Christophe
JC
Gautier
Clément
C
Robin
Anne-Claire
AC
Gmouh
Said
S
Berka
Vladimir
V
Tsai
Ah-Lim
AL
Blanchard-Desce
Mireille
M
Slama-Schwok
Anny
A
D001665:Binding Sites; D001667:Binding, Competitive; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry
D005415:Flavins; D006418:Heme; D009249:NADP; D052250:Nitric Oxide Synthase Type III