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

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 (k=3.9(4) s) lower than that of wt SmNir (k=240(50) s). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the NH…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the NH…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.

A three-domain copper-nitrite reductase with a unique sensing loop

“…The T1Cu N ! T2Cu electron-transfer pathway of TsNirK consists of the well characterized Cys-His bridge observed in all NirKs reported so far (Brenner et al, 2009;Cristaldi et al, 2018;Leferink et al, 2011;Strange et al, 1999). Electron delivery towards the T2Cu active site through the Cys-His bridge has been demonstrated to be regulated by the so-called 'sensing loop', which harbours an Asp CAT residue essential for catalysis (Boulanger et al, 2000;Kataoka et al, 2000;Strange et al, 1999).…”

Studying Electron Transfer Pathways in Oxidoreductases

“…). This feature allows simulating each spectrum separately, and also to quantify T1 and T2 concentrations; EPR parameters can be found in (Cristaldi et al 2018). Upon incubation with sodium ascorbate (E ~ 0 mV), T1 becomes EPR silent, as it is reduced to Cu 1+ (d 10 , S= 0), whereas a fraction of T2 remains in the Cu 2+ oxidation state (Figure 4, spectrum B).…”

“…Second, the two copper centers show similar relaxation rates. Therefore, to evaluate the integrity of the ET pathway in NirK, we exploited the pH dependence of its catalytic activity (Cristaldi et al, 2018). NirK presents maximal activity at pH 5-6 and no activity at pH >8, in which the dependence with pH could be attributed the N d1 H…O=C hydrogen bond present in the T1-T2 ET pathway (Figure 1).…”

“…As shown in Figure 1, this pathway has two subpaths, one that involves a pure covalent chemical pathway (highlighted in blue) constituted by the protein backbone and the Cys and His side chains, and another one that is a mixed chemical pathway constituted by a His-N d1 H…O=C-Cys hydrogen bond that partially shortcuts the T1-T2 bridging covalent link (highlighted in green, Figure 1). We constructed two variants of S. meliloti NirK, H171D and C172D (Note: H171D and C172D are two protein variants where the residues histidine 171 and cysteine 172 were changed to aspartic acid), and their kinetic, structural, and spectroscopic properties were compared with those of the wild type enzyme (Cristaldi et al, 2018). The H171D variant (Figure 7) contains both T1 and T2 only connected by the pure covalent subpath and, even though it retains its ability to bind the substrate, it is unable to catalyze nitrite reduction.…”

“…Readers interested in reading about alternative applications of this technique related to ET studies can see reference (Davidson, 2008). (Cristaldi et al, 2018). The N d1 H…O=C hydrogen bond is depicted in red.…”