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

CRISTALDI, JULIO C.; GÓMEZ, MARÍA C.; GONZÁLEZ, PABLO J.; FERRONI, FELIX M.; DALOSTO, SERGIO; RIZZI, ALBERTO C.; RIVAS, MARÍA G.; BRONDINO, CARLOS D.

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Año: 2018 vol. 1862 p. 752 - 760

0304-4165

TheCys-His bridge as electron transfer conduit in the enzymatic catalysis ofnitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti2011 (SmNir) was evaluated by site-directedmutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopicmeasurements as well as computational calculations. The kinetic, structural andspectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with thewild type enzyme. Molecularproperties of H171D and C172D indicate that these point mutations have not visible effects on the quaternarystructure of SmNir. Both variantsare catalytically incompetent using the physiological electron donorpseudoazurin, though C172D presents catalytic activity with the artificialelectron donor methyl viologen (kcat= 3.9(4) s-1) lowerthan that of wt SmNir (kcat=240(50) s-1). QM/MM calculations indicate that the lack of activityof H171D may be ascribed to the Nd1H?O=C hydrogen bond that partially shortcuts the T1-T2bridging Cys-His covalent pathway. The role of the Nd1H?O=C hydrogen bond in the pH-dependent catalyticactivity of wt SmNir is also analyzedby monitoring the T1 and T2 oxidation states at the end of the catalyticreaction of wt SmNir at pH 6 and 10by UV-vis and EPR spectroscopies. These data provide insight into how changesin Cys-His bridge interrupts the electron transfer between T1 and T2 and howthe pH-dependent catalytic activity  ofthe enzyme are related to pH-dependent structural modifications of the T1-T2bridging chemical pathway