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2 results on '"Arg375"'

1. Arg375 tunes tetrahydrobiopterin functions and modulates catalysis by inducible nitric oxide synthase

Author

Wang, Zhi-Qiang, Tejero, Jesús, Wei, Chin-Chuan, Haque, Mohammad Mahfuzul, Santolini, Jerome, Fadlalla, Mohammed, Biswas, Ashis, and Stuehr, Dennis J.

Subjects
*TETRAHYDROBIOPTERIN, *NITRIC-oxide synthases, *CATALYSIS, *PTERIDINES, *HYDROGEN bonding, *ARGININE, *MUTANT proteins
Abstract

Abstract: NO synthase enzymes (NOS) support unique single-electron transitions of a bound H4B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H4B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H4B ring. In order to understand the role of this residue in modulating the function of H4B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H4B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O2. Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H2O2-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H4B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H4B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H4B during steady state NO synthesis and H2O2-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H4B and is important in controlling the catalytic function of NOS enzymes. [Copyright &y& Elsevier]

Published
2012
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2. Arg375 tunes tetrahydrobiopterin functions and modulates catalysis by inducible nitric oxide synthase

Author

Mohammed Fadlalla, Ashis Biswas, Jérôme Santolini, Zhi Qiang Wang, Chin Chuan Wei, Dennis J. Stuehr, Mohammad Mahfuzul Haque, Jesús Tejero, Kent State University, Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, affiliation inconnue, Southern Illinois University [Edwardsville] ( SIUE ), Stress Oxydants et Détoxication ( LSOD ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), National Institutes of Health Grants GM51491 and CA53914, American Heart Association Beginning Grant-in-aid 0565297B, KSU Farris Innovation Award, KSU Tuscarawas Faculty Professional Development Release Time Award, Southern Illinois University [Edwardsville] (SIUE), Stress Oxydants et Détoxication (LSOD), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
MECHANISM, Nitric Oxide Synthase Type II, Biochemistry, [ CHIM ] Chemical Sciences, Protein Structure, Secondary, Hydroxylation, chemistry.chemical_compound, 0302 clinical medicine, ELECTRON-TRANSFER, Pterin, Heme, 0303 health sciences, Tetrahydrobiopterin, biology, Nitric oxide synthase, BINDING-SITE, Single turn over, Protein Binding, inorganic chemicals, Stereochemistry, OXYGENASE DOMAIN, Arginine, Redox, Catalysis, Article, Cofactor, Ferrous, NO, Inorganic Chemistry, 03 medical and health sciences, HEME-DIOXY REDUCTION, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Arg375, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, SINGLE-TURNOVER, Hydrogen Bonding, Biopterin, STOPPED-FLOW ANALYSIS, HYDROXY-L-ARGININE, RADICAL FORMATION, chemistry, Mutagenesis, Site-Directed, biology.protein, Steady state (chemistry), NEURONAL NO SYNTHASE, Protein Multimerization, 030217 neurology & neurosurgery, Midpoint potential
Abstract

International audience; NO synthase enzymes (NOS) support unique single-electron transitions of a bound H(4)B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H(4)B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H(4)B ring. In order to understand the role of this residue in modulating the function of H(4)B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H(4)B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O(2). Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H(2)O(2)-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H(4)B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H(4)B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H(4)B during steady state NO synthesis and H(2)O(2)-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H(4)B and is important in controlling the catalytic function of NOS enzymes.

Published
2012

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