Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase.
NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour.
Tejero
Jesús
J
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Biswas
Ashis
A
Haque
Mohammad Mahfuzul
MM
Wang
Zhi-Qiang
ZQ
Hemann
Craig
C
Varnado
Cornelius L
CL
Novince
Zachary
Z
Hille
Russ
R
Goodwin
Douglas C
DC
Stuehr
Dennis J
DJ
D001426:Bacterial Proteins; D001692:Biological Transport; D002384:Catalysis; D004583:Electrons; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008652:Mesoporphyrins / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D008652:Mesoporphyrins; D006418:Heme; C031468:mesoporphyrin IX; D052248:Nitric Oxide Synthase Type I