Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

• Published in: Biochimica et biophysica acta Vol. 1862; no. 3; pp. 752 - 760
• Main Authors: Cristaldi, Julio C., Gómez, María C., González, Pablo J., Ferroni, Felix M., Dalosto, Sergio D., Rizzi, Alberto C., Rivas, María G., Brondino, Carlos D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2018
• Subjects: catalytic activity; Copper; Denitrification; electron paramagnetic resonance spectroscopy; electron transfer; Electron transfer pathway; Ensifer meliloti; enzyme activity; Enzyme mechanism; hydrogen bonding; monitoring; nitric oxide; Nitrite reductase; nitrites; oxidation; paraquat; point mutation; Sinorhizobium meliloti 2011; site-directed mutagenesis; spectral analysis; ultraviolet-visible spectroscopy; Rs; wt SmNir; Af; Nitrite reductase; MV; QM/MM; Sinorhizobium meliloti 2011; SmPaz; H171D; Electron transfer pathway; Enzyme mechanism; Ax; Denitrification; C172D; Copper
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2017.10.011

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s−1) lower than that of wt SmNir (kcat=240(50) s−1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the Nδ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway. [Display omitted] •Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase•Site-directed mutagenesis, spectroscopic, and computational methods•Protein variants are catalytically incompetent towards physiological electron donor.•Changes in the Cys-His bridge interrupt the T1–T2 electron transfer.•The Nδ1H…OC hydrogen bond is essential for catalysis.