Fundamental studying of laccases from basidiomycetes, namely biochemical and electrochemical studyin

1
Fundamental studying of laccases from
basidiomycetes, namely biochemical and
electrochemical studying for knowledge of the
real mechanism of their functioning
2
Crystal structure of the laccase(Trametes
versicolor)
Laccase (p-diphenoloxygen oxidoreductase, EC
1.10.3.2) is multi-copper oxidase that catalyzes
the oxidation of various aromatic compounds and
also some inorganic compounds with the
concomitant reduction of dioxygen to water. The
laccases can be divided into two categories,
plant and fungal, although diphenol-oxidizing
enzymes which are thought to be laccases have
also been identified in insects and eubacteria.
Due to its biochemical, catalytical and
electrochemical properties laccase has gained
considerably interest because of its potential
biotechnological applicability. The quaternary
structure of laccase has been solved only for a
limited number of enzymes, e.g. Coprinus
cinereus, Melanocarpus albomyces, Pycnoporus
cinnabarinus, and Trametes versicolor. It was
shown that laccases have a minimum of one
mononuclear copper site containing one type-1 Cu,
and a trinuclear copper site containing one
type-2 Cu and two type-3 Cu. Oxidation of simple
organic substrates occurs via a ping-pong type
mechanism. Substrates are oxidized near the
mononuclear site, and the electrons are
transferred to the trinuclear site, where the
molecular oxygen is reduced. Neither the electron
transfer mechanism nor the oxygen reduction to
water is fully understood. Nevertheless, a number
of mechanistic schemes satisfactory describing
the set of kinetic and structural data has been
proposed.

3
Questions 1. What is the first electron
acceptor in laccase in case of an homogeneous
reaction and in the time of the electrochemical
reaction (heterogeneous), and what kind of site,
T1, T2 or T3 ??? 2. Which substrate is the
first for laccase in the catalytical cycle
oxygen or donor of electrons ??? 3. Is DET
possible for laccase at aerobic and especially
anaerobic conditions using different kinds of the
electrodes, for example gold or HOPG electrodes
??? 4. What are the values of the standard redox
potentials of T1, and especially T2 and T3 sites
for laccases from different sources??? Are those
values the same at aerobic and anaerobic
conditions ???
4
Basic biochemical properties of the laccases from
different source
5
Spectral studying of the laccases from Trametes
ochracea (a), Trametes hirsuta (b), Coriolopsis
fulvocenerea (c) and Cerrena maxima (d)
Absorbance spectra of the extra cellular fungal
laccases
EPR-spectra of the laccases
c
b
b
a
c
d
d
b
Absorbance
a
a
c
Absorbance
20 mT
d
Emission fluorescence spectra (I) and excitation
fluorescence spectra (II) of the laccases
(I)
(II)
Wavelength, nm
Relative intensity of fluorescence
Wavelength, nm
350 nm
520 nm
280 nm
400 nm
6
Redox potential measurement of T1 site Trametes
hirsuta (I) and Trametes ochracea (II) laccases
by potentiometric redox titration using
K4Mo(CN)8 and K3Mo(CN)8 as the mediators
A
2
A
1
1
native enzyme
3
1
native enzyme
1
lg(A/(A0-A)
lg(A/(A0-A)
2
5
4
3
4
2
3
2
Potential, mV
4
Potential, mV
3
5
4
II
I
?, nm
?, nm
7
DET (bioelectrocatalytic reduction of oxygen) for
laccase fromTrametes ochracea (A), Coriolisimus
fulvocinerea (B), and Cerrena maxima
(C)spectroscopic graphite electrode (SGE)(50 mM
citrate-phosphate buffer saturated oxygen pH 3.0
ionic strength - 0.1 M NaClO4 scan rate 10
mVs-1 start potential - 900 mV )
without laccase
without laccase
A
C
B
without laccase
? / ?A
B
with laccase
with laccase
with laccase
Potential, mV (vs. Ag/AgCl)
Potential, mV (vs. Ag/AgCl)
Potential, mV (vs. Ag/AgCl)
8
pH activity dependence of electroreduction of
oxygen by laccases from different source
(spectroscopic graphite)RV Rhus vernicifera,
TH Trametes hirsuta, TZ T. zonatus, CU
Cerrena unicolor, CM C. maxima, CF
Coriolisimus fulvocinerea
Basic electrochemical properties of the laccases
from different source
CU
TH
Activity of laccase, µA
RH
CM
TO
CF
pH
9
Bioelectrocatalytic reduction of oxygen on the
Trametes hirsuta laccase modified SGE electrode
50 mM citrate-phosphate buffer saturated oxygen
pH 3.0 ionic strength - 0.1 M NaClO4 scan rate
10 mVs-1 start potential - 900 mV
pH activity dependence of Trametes hirsuta
laccase (1) The activity of laccase in
homogeneous solution measured by K4Fe(CN)6, one
unit of activity is defined as change in A410/min
per mg of protein. (2) The electrocatalytic
reduction of oxygen at the laccase modified
spectroscopic graphite electrode. One unit of
activity is the current (µA) of the electrode.
The laccase activity was determined as an oxygen
reduction current of laccase modified electrodes
at a potential of 350 mV vs. NHE electrode at a
saturation part in the cyclic voltammograms
without laccase
Activity
? (?A)
with laccase
E (mV) vs. NHE
pH
10
Osteryoung square wave voltammograms recorded
with HOPG electrode with and without T. hirsuta
laccase entrapped under a dialysis membrane at
the electrode surface 0.1 M phosphate buffer pH
6.5 S. W. Amplitude 10 mV Frequency 10 Hz.
Cyclic voltammograms recorded with HOPG electrode
with and without the T. hirsuta laccase entrapped
under a dialysis membrane at the electrode
surface 0.1 M citrate-phosphate buffer pH 3..0,
scan rate 10 mV/s
without laccase
with laccase
? (?A)
? (?A)
without laccase
with laccase
E (mV) vs. NHE
E (mV) vs. NHE
11
The steady state potential of a gold electrode
with laccase, (0.1 M citrate-phosphate buffer pH
3.0)
12

Cyclic voltammograms of laccase in the gold
capillary electrode The experiments were
performed in 0.1 M phosphate buffer pH 6.5, in
(A) and (B) the solution containing 10 mg/ml of
T. hirsuta laccase in 0.1 M phosphate buffer pH
6.5 (A) Cyclic voltammogramms recorded at
different scan rates with the T. hirsuta laccase
in 0.1 M phosphate buffer pH 6.5, start potential
0 mV vs. NHE (B) Cyclic voltammograms at 10
mV/s in a broader potential window, start
potential 1030 mV vs. NHE, 1 - control
measurement in the absence of the laccase, 2
with laccase. Insert The dependence of anodic
and cathodic peak current vs. square root of the
potential scan rate (V/s).
B
A
50 mV/s
25 mV/s
2
10 mV/s
1
5 mV/s
? (?A)
ip, a
?p (?A)
ip, c
V1/2
E (mV) vs. NHE
13
Redox titration of Trametes hirsuta laccase using
the spectroelectrochemical gold capillary
cell (A) Redox titration of Trametes hirsuta
laccase using the spectroelectrochemical gold
capillary cell. 0.1 M phosphate buffer pH 6.5.
(E) and (1) to (5) are absorbance spectra of
initially injected enzyme and at applied
potentials 1030, -20, 530, 830, and 1030 mV,
respectively. (B) Spectroelectrochemical
titration curves reflecting the dependence of
optical absorbance of the laccase solution at 614
nm vs. the applied potential. Titration curves
recorded by changing the applied potential (1)
from -20 to 1030 mV and (2) from 1030 to -20 mV,
respectively.
1
A
B
2
E
E0? 330 mV
T1
4
Absorbance
site ?
Absorbance (614 nm)
2
3
E0? 865 mV
1
gold electrode
2
?, nm
E (mV) vs. NHE
14

• Conclusions
• Homogeneous preparation of laccases have been
  obtained and characterised. The key biochemical
  properties such as molecular mass, pH-optimum,
  stability, copper content, potential of the T1
  site, and specific activity were determined. It
  was shown that our laccase are a high redox
  potential enzymes with the biochemical
  characteristics close to other blue high
  potential fungal laccases.
• It was shown that the laccase solution contained
  some part of the native enzyme with the copper in
  active site in its reduced state (data not
  shown).
• The electrochemistry of five laccases from
  basidiomycetes adsorbed on graphite electrode was
  investigated. It is concluded that the T1 site of
  the enzymes is the first electron acceptor, both
  in solution (homogenous case) and when the
  laccase is adsorbed on the surface of the
  graphite electrode (heterogeneous case) under
  aerobic conditions. Moreover, from comparing of
  the biochemical and electrochemical results it is
  suggested that the reaction mechanism of oxygen
  reduction catalysed by laccase in homogeneous (in
  solution) and heterogeneous (adsorbed on the
  carbon surface) conditions are similar in many
  respects.
• The electrochemistry of T. hirsuta laccases on
  graphite and gold electrodes was investigated in
  detail. It was shown the possibility of DET for
  laccase under anaerobic and aerobic conditions on
  SGE, HOPG, and gold electrodes.
• It was found that the mechanism of electron
  transfer between laccase and the two different
  electrodematerials (carbon and gold) was
  concluded to be totally different. At carbon
  electrodes DET is observed between the surface of
  the electrode and the T1 site. To explain DET of
  laccase on gold a gating of the ET process
  between the surface of gold and the T1 site is
  proposed. The nature of the gating redox centre
  was not determined, but it could be an amino
  acid, an S-S bridge or the T2/T3 copper centre.

15
Swedish team
Russian team
1. Professor Alexander Yaropolov 2. Sergey
Shleev, Ph. D. 3. Olga Morozova, J.
researcher 4. Anna Khalunina, Ph. D. Student

• Professor Lo Gorton
• 2. Assoc. Professor Tautgirdas Ruzgas
• 3. Andreas Christenson, Ph. D. Student

Acknowledgements The work was supported by
INCO-Copernicus grant (contract No.
ICA2-CT-2000-10050)