Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis,spectroscopic, and computational methods |
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| Authors: | Julio C. Cristaldi María C. Gómez Pablo J. González Felix M. Ferroni Sergio D. Dalosto Alberto C. Rizzi María G. Rivas Carlos D. Brondino |
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| Affiliation: | 1. Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral/CONICET, S3000ZAA Santa Fe, Argentina;2. Instituto de Física del Litoral, CONICET-UNL, Güemes 3450, S3000ZAA Santa Fe, Argentina |
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| Abstract: | 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…O | |
| Keywords: | MV methyl viologen QM/MM quantum mechanics/molecular mechanics C172D H171D Nitrite reductase Copper Denitrification Enzyme mechanism Electron transfer pathway |
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