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

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• 作者：Zhi-Qiang Wang^a ; ^{zwang3@kent.edu} ; Jesú ; s Tejero^b ; Chin-Chuan Wei^c ; Mohammad Mahfuzul Haque^b ; Jerome Santolini^d ; Mohammed Fadlalla^b ; Ashis Biswas^b ; Dennis J. Stuehr^b ; ^{stuehrd@ccf.org}
• 关键词：NO ; Single turn over ; Arg375 ; Nitric oxide synthase ; Tetrahydrobiopterin ; Midpoint potential
• 刊名：Journal of Inorganic Biochemistry
• 出版年：2012
• 期刊代码：53_01620134
• 类别：ch
• 出版时间：March, 2012
• 卷：108
• 期：Complete
• 页码：203-215
• 文件大小：1512 K

摘要

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.