PROJECT PROPOSAL
for applicants for Ph.D. fellowships
supervisor:
institution:
contact:
CV:
Csaba BAGYINKADSB, DSMMS, Ph.D., D.Sc.
Institute of Biophysics
bagyinka.csaba@brc.mta.hu
http://www.brc.hu/file/cv/bf_bagyinka_csaba_en.pdf
project title:
AUTOCATALYTIC ENZYME REACTION
PROJECT SUMMARY
Hydrogenases are metalloenzymes that catalyze the reversible oxidation and reduction of molecular
hydrogen, H2 ⇌ 2p+ + 2e-. There are two autocatalytic reaction steps in the H2-oxidizing reaction
of hydrogenase. One autocatalytic reaction occurs when the enzyme is mainly in inactive forms. The
most obvious sign of this autocatalytic step is that the reaction starts from distinct points. The
reaction-diffusion front is direct evidence of an autocatalytic reaction. The second autocatalytic step
is located in the enzyme cycle. Its presence is supported by the facts that the Ni-R form is
exclusively diamagnetic and the steady-state concentration of the product of the H2-oxidizing
reaction displays a strong enzyme concentration dependence. The student will study the details of
these autocatalytic reaction steps; identify their localization in the reaction cycle, identify the
autocatalyst etc. using different biochemical and spectroscopic methods.
BACKGROUND OF THE STUDY
Hydrogenases are metalloenzymes that
catalyze the reversible oxidation and reduction
of molecular hydrogen.The active centrum of
the enzyme is a very special [Ni-Fe] centernad
it also contains three [FeS] clusters. Their
hydrogen oxidation (hydrogen uptake) activity is
important, because hydrogenase could replace
the expensive platinum electrodes in fuel cells.
Previous experimental results and theoretical
considerations led to the conclusion that there
are two autocatalytic reaction steps in the H2oxidizing reaction of hydrogenase. One
autocatalytic reaction occurs when the enzyme
is mainly in inactive forms (Ni-A and/or Ni-B).
This finding is supported by the special patterns
of H2 oxidation in a thin-layer reaction chamber
and by the special, long lag phase observed.
The autocatalytic step takes place between two
enzyme forms, one of which also interacts
directly with the terminal electron acceptor, i.e.
the autocatalyst is an enzyme form in which
[FeS]distal is reduced. During the autocatalytic
interaction, an electron is passed from the
autocatalyst to the other enzyme form, i.e.
inactive hydrogenase serves as a substrate in
this autocatalytic step for another, activated
hydrogenase molecule.
The most obvious sign of this autocatalytic step
is that in a thin layer reaction chamber the
reaction starts from distinct points, spreading
out continuously until the whole mixture
becomes blue. New spots may appear and
grow during the reaction. The spots eventually
collide and fuse. Within experimental error, the
spots grow at constant velocity during the
experiment (Fig. 1). The reaction-diffusion front
is direct evidence of an autocatalytic reaction.
The second autocatalytic step is located in the
enzyme cycle. Its presence is supported by the
facts that the Ni-R form is exclusively
diamagnetic and the steady-state concentration
of the product of the H2-oxidizing reaction (i.e.
reduced benzyl viologen) displays a strong
enzyme concentration dependence. This
enzyme concentration dependence does not
depend on the “active-inactive” states of the
enzyme, and remains valid in consecutive H2oxidizing - proton-reducing reaction series
performed on the same sample (Fig. 2). This
autocatalytic step involves an enzyme-enzyme
interaction; both interacting enzyme forms
should participate in the catalytic cycle of the
enzyme. Since the reduction of all [FeS]
clusters would also be possible in a nonautocatalytic reaction, we hypothesize a small
conformational change in the enzyme,
catalyzed by the autocatalyst, which removes a
block in the electron flow in either the
[NiFe]/[FeS]proximal
or
the
[FeS]proximal/[FeS]distal reaction step, or
removes a blockade of the penetration of
gaseous H2 from the surface to the [NiFe]
cluster.
A number of autocatalytic enzyme reactions are
known. In the peroxidase system, the inorganic
reaction mechanism includes autocatalysis; the
enzyme merely catalyzes certain of the reaction
steps. In autophosphorylation, the inactive
enzyme is the substrate of its active form, which
transforms the inactive form into an active one.
This is very similar to the first type of
autocatalytic reaction in hydrogenase.
Furthermore, in various allosteric enzyme
reactions,
the
enzyme
is
simply
activated/inhibited by its substrates or products.
In the second case, when the autocatalytic
reaction occurs in the enzyme cycle,
hydrogenase participates in a unique type of
autocatalysis, where the autocatalyst enzyme
form interacts with another enzyme form,
resulting in a probably small conformational
change in the enzyme-substrate. This scheme
is very similar to the prion reactions, but
significantly different from the other known
autocatalytic enzyme reactions described
above. As far as we are aware, such a
phenomenon has not yet been observed
experimentally previously for an enzyme. The
main characteristic of this kind of enzyme
reaction that there is no steady state kinetics in
the enzyme reaction, i.e. the concentrations of
different conformers in the enzyme catalytic
cycle cannot be assumed to be constants ever.
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project
RELEVANT RESEARCH IN THE HOST
LABORATORY
Although there were several indications in the
literature, our laboratory was pioneering in
discovery of the autocatalytic reaction in the
hydrogenase reaction cycle. We have identified
the autocatalyst as an enzyme form in which
the [FeS]distal is reduced. Recently we are
trying to identify the exact enzyme form and the
reaction steps where the autocatalytic reaction
takes
place.
Preliminary
temperature
dependent
activity
and
spectroscopic
measurements were performed, and deviations
from the “normal” enzyme behavior were
observed. Under specific conditions an
oscillation can also be observed in the reaction
but the concrete details of this phenomenon are
not known. We also build mathematical models
in order to investigate the enzyme reaction and
evaluate and model the experimental results.
The autocatalytic reaction of hydrogenase in thin layer.
The reaction-diffusion fronts are clearly observable.
SPECIFIC AIMS
Detect the suspected conformational change
during the autocatalytic reaction by different
spectroscopic methods.
Experimentally clarify the parameters resulting
oscillatory phenomena in hydrogenase reaction.
Find a mathematical model for the oscillations
in the hydrogenase reactions.
Find the proper experimental conditions where
the different hydrogenase conformations can be
crystallized.
Since the object of the study – the
hydrogenase – is always needed a never
ending task is to produce pure enzyme for
measurements.In order to achieve this goal
the student should be familiar (or able to
learn it) with:
o Cultivation of photosynthetic bacteria
o Different protein purification and
analysis methods.
Different methods to determine the activity of
hydrogenase
Anaerobic spectroscopic methods.
Building and solving kinetic models using
MATLAB computational tools.
SUGGESTED READINGS
Time course of H2 oxidation in the modified thin-layer
reaction chamber. Since the time courses in the
experiment exhibited different lag phases, in this Figure
the graphs have been shifted to uniform lag phase (t=0).
Experimental conditions: enzyme concentration varying
from 4 nM to 1060 nM, oxidized benzyl viologen
concentration 400 µM.
Pandelia ME, et al.: Intermediates in the Catalytic Cycle
of [NiFe] Hydrogenase: Functional spectroscopy of the
active site. Chemphyschem. 11:1127-1140(2010)
Bagyinka Cs: How does the hydrogenase enzyme work?
Int. J. Hydr. Energy, 39:18521-18532(2014)
MATERIAL AND METHODS
SNAPSHOTS FROM THE HOST LABORATORY
Significant publications
Bagyinka C, et al.: Autocatalytic oscillations in the early phase of the photoreduced methyl viologen-initiated fast kinetic
reaction of hydrogenase. J. Biol. Chem., 278:20624-20627(2003)
Ősz J, et al.: An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the
special characteristics of the enzyme reaction. Biophys. J., 89:1984-1989(2005)
Ősz J, et al.: Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration
dependence of the activity of hydrogenase uptake. Biophys. J., 89:1957-1964(2005)
Bodó G, et al.: Concentration-dependent front velocity of the autocatalytic hydrogenase reaction. Biophys. J., 96:49764983(2009)
Bankó S, et al.: The autocatalytic step is an integral part of the hydrogenase cycle. Biochim. Biophys. Acta, 1834:658664(2013)
Bagyinka C, et al.: Oscillating hydrogenase reaction. Int. J. Hydogen Energy, 39:18551-18555(2014)
Bagyinka Cs: How does the hydrogenase enzyme work? Int. J. Hydr. Energy, 39:18521-18532(2014)
Representative recent research grants
”Investigation of the autocatalytic enzyme reaction of hydrogenase” (OTKA, 2005-2009)
”The oscillating hydrogenase reaction” (OTKA, 2011-2016)
Some of the latest students in the laboratory
Ősz J, Ph.D., 1999-2005; “The autocatalytic reaction of hydrogenase”
Branca RMM, Ph.D., 2002-2008; “Structure and function of a novel cytochrome c4 from the purple photosynthetic
bacterium Thiocapsa roseopersicina”
Pankotai-Bodó G, Ph.D., 2001-2009; "Investigation of the autocatalytic reaction of hydrogenase”
Bankó S, Ph.D., 2009-recent; “To be announced”
Janovics Zs, Ph.D., 2009-recent;“To be announced”
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project