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

Abstract

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.

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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
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