Study of physiological copper nitrite reductase mediators towards biosensor development

CINTIA SOLEDAD RAMIREZ; PABLO JAVIER GONZÁLEZ; CARLOS DANTE BRONDINO; FELIX MARTÍN FERRONI

Paraná

Congreso; 54th Annual Meeting Argentine Society for Biochemistry and Molecular Biology. LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología molecular.; 2018

SAIB

Denitrifiers are a good source of enzymes that are useful for the development of biodevices for N-compounds sensing in environmental samples. Particularly, nitrite reduction is the way to enter into the denitrification pathway mediated by nitrite reductase enzyme. Two kind of nitrite reductases can be found in nature: copper- and cytochrome cd1-containing nitrite reductases, NirK and NirS, respectively. NirKs receive the electrons from azurins, pseudoazurins and cytochrome c550, depending on the microorganism. Usually, green NirKs receive electrons from pseudoazurins while blue Nirks from azurins or c-type cytochromes. Nevertheless, Nirks rarely interacts with electrodes directly. Hence, we need a physiological mediator that is able to interact with the electrode surface in a biosensor. Moreover, the best mediator-enzyme couple has to be experimentally determined for the design of the biosensor.We have cloned, expressed and purified several physiological mediators for NirKs: a green pseudoazurin from Sinorhizobium meliloti 2011 (SmAzu2) and a cytochrome c550 fom Bradyrhizobium japonicum USDA110 (BjCycA). We report here biochemical and electrochemical studies of the interaction of these mediators and several NirKs: a green NirK (SmNirK), a blue NirK (BjNirK) and a thermotolerant NirK from Thermus scotoductus SA-01 (TsNirK).The reoxidation of the mediator under argon atmosphere was spectrophotometrically monitored in a continuous assay in a sealed cuvette. All the electrochemical studies were performed on a 4-4´-dithiopyridine-sensibilized gold electrode. Cyclic voltammetry and chronoamperometry assays were performed.