Nitrite Reductase, an enzyme for which kinetics are different at rest than during turn over.

Lukasz Krzeminski 1Lionel Ndamba 2Gerard W. Canters 2Thijs J. Aartsma 2Stephen D. Evans 1Lars Jeuken 1

1. University of Leeds (UOL), Woodhouse Lane, Leeds ls2-9jt, United Kingdom
2. Leiden Univ., Leiden, Netherlands


A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turn-over rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Förster Resonance Energy Transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 mM) resulting in an inhibition effect at low pH and catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turn-over conditions compared to the enzyme at rest, with an exception at low pH / nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper centre due to a rate-limiting protonation step of residues in the enzyme’s active site, gating the intramolecular electron transfer.


Related papers
  1. Electrodes for integral membrane enzymes
  2. Electron and Proton Transfer Across Biological Membranes, Measured Using Vesicles Immobilised on Electrode Surfaces.

Presentation: Short communication at SMCBS'2011 International Workshop, by Lukasz Krzeminski
See On-line Journal of SMCBS'2011 International Workshop

Submitted: 2011-07-13 16:14
Revised:   2011-07-13 16:32