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Updated: Aug 6, 2026

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
An apparently allosteric effect involving N2O with the nitrous oxide reductase from Wolinella succinogenes
C Zhang1, A M Jones, T C Hollocher
1Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Abstract:
It was shown that kcat for the benzyl viologen cation (BV+)-N2O oxidoreductase activity of nitrous oxide reductase from Wolinella succinogenes was 2-3 times greater at high N2O concentrations than at low. This effect of N2O on kcat exhibited a titration curve implicating a single secondary binding site for N2O with a Kd of 130-200 microM (Km with respect to N2O is about 2.5 microM). This work represents the first evidence of an apparently allosteric kinetic effect among nitrous oxide reductases. Its possible cause is discussed. BV+ was generated in these kinetic studies by addition of sub-stoichiometric amounts of dithionite. This means of reduction proved to be superior to the photochemical generation of BV+ that had been used previously with the enzyme. Mass spectrometric measurements suggested that the M(r) of the subunit of the enzyme is about 95,500 rather than 88,000.
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