Role of arginine guanidinium moiety in nitric-oxide synthase mechanism of oxygen activation.
Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate L-Arg first to N(omega)-hydroxyl-L-arginine and then to L-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the L-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of L-Arg analogues exhibiting a wide range of guanidinium pK(a) values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pK(a) value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.
Giroud
Claire
C
Laboratoire Stress Oxydants et Detoxication, Commissariat à l'Energie Atomique Saclay, Institut de Biologie et de Technologies de Saclay, 91191 Gif-sur-Yvette Cedex, France.
Moreau
Magali
M
Mattioli
Tony A
TA
Balland
Véronique
V
Boucher
Jean-Luc
JL
Xu-Li
Yun
Y
Stuehr
Dennis J
DJ
Santolini
Jérôme
J
D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D019791:Guanidine / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D026361:Reactive Nitrogen Species / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D002956:Citrulline; D009569:Nitric Oxide; D006418:Heme; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D019791:Guanidine; D010100:Oxygen