Development of rapid freeze-quench techniques for studying
intermediates in enzymatic reactions of denitrification
PhD student: Ir. F.G.M.Wiertz Phone: +31 15 2788475
e-mail: [email protected]
Promotor:
Prof.dr. S. de Vries
Delft University of Technology, Department of
Institute:
Biotechnology, Enzymology group
Project term: August 2000 – August 2004
Financed by: FOM
Description
Denitrification is the process in which Bacteria
and Archaea use nitrate (NO3-) as terminal
electron acceptor for energy production
instead of molecular oxygen (Fig. 1).
Our research concentrates on the enzymes
nitrite reductase (NIR) and nitric oxide
reductase (NOR) from Paracoccus
denitrificans. Little is known about the
intermediates formed during the reduction of
the substrate for NIR and even less for NOR
because the reactions are so fast.
To determine the structure of these
intermediates one needs to stop the reaction
between enzyme and substrate in less then 1
ms after mixing. For the study of
intermediates with UV-Vis or fluorescence
spectrophotometry commercial equipment
(stopped-flow devices) is available with deadtimes of around 1 ms. If other measuring
techniques are necessary, like EPR for the
study of the metallo-proteins in this research,
then freeze-quench devices with dead times
of around 5 ms are available because the
samples need to be frozen for further sample
handling. The sample is frozen in coldisopentane (aprox. –140 oC) but becomes
highly diluted which is a drawback if EPR
measurements are done.
In our group a micro mixer was developed
that has a mixing time of less then 10 µs. A
jet leaves the mixer with a speed of 200m/s. It
has a diameter of 20 µm which is thin enough
to freeze it in approximately 10-40 µs at a
rotating cold plate (Fig. 2). The sample has a
reaction time corresponding to the time of
flight. This is the time taken by the jet
travelling from mixer to cold plate. A thin
sample layer is sprayed on the rotating cold
NAR
NO3NO3-
NO2-
pseudo-azurin
cytochrome c 551
periplasm
NIR
bc1
QH2
NO2 -
NO
cytoplasm
DH
NO2Q
cytoplasmic
membrane
C 552
NOR
NO
outermembrane
N2O
bc1
QH2
N 2 OR
pseudo-azurin
cytochrome c 551
N2 O
N2
Fig.1 Schematic overview of denitrification in
proteobacterea. Nitrate, nitrite, NO an N2O all
function as final electron acceptors.
Fig. 2 Freeze-quench setup. The cold plate
and mixer are shown. The coldplate is
cooled with N2 (l) down to 77K. The spray
head can move up and down to spray a thin
layer on the cold plate.
plate. Because of the high speed of the jet the minimum time of flight is 25 µs. This makes it
theoretically possible to freeze-quench a sample within 40 µs. The samples can be used for
UV-Vis at 77K and EPR measurements down to 4.2K. Not only the freeze-quench time is
improved with this set-up by a factor of 100, but also the fact that the sample is no longer
frozen in cold isopentane is a great advantage. This set-up is being tested and further
improved in this project and later several enzymes will be studied to detect novel reaction
intermediates.
References
Wasser, I., de Vries, S., Moënne-Loccoz, P., Schröder, I. And Karlin, K.D. (2002). Nitric oxide
in biological denitrification: Fe/Cu Metalloenzyme and metal complex NOx redox chemistry.
Chem.Rev. 102, 1201-1234
Ballou, D.P. and Palmer, G.A. (1974). Practical rapid quenching instrument for the study if
reaction mechanisms by electron paramagnetic resonance spectroscopy. Anal. Chem. 46,
1248-1253