Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays

摘要

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 isable to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensivelycharacterized from the spectroscopic and structural points-of-view, some of its kinetic aspectsare still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in asystematic manner using two different spectrophotometric assays carried out in the presence ofdifferent redox mediators and a direct electrochemical approach. Solution assays have provedthat the specific activity of ccNiR decreases with the reduction potential of the electronic carriersand ammonium is always the main product of nitrite reduction. The catalytic parameters werediscussed on the basis of the mediator reducing power and also taking into account the locationof their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron deliveringfrom reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimatedkinetic constants are apparent only. Nevertheless, this limitation could be overcome by using adirect electrochemical approach which shows that the binding affinity for nitrite decreases whilstturnover increases with the reductive driving force.