Redox potentials of fungal laccases: their measurement via changes in optical absorption of the t1
copper site following exposure to various redox couples
Taryn Dooms1, Jenna Zimmerman2, and Brian W. Gregory3, Ph.D.
1
Tuskegee University, 70803; email: [email protected]
2
Samford University, Birmingham, AL 35229; email: [email protected]
3
Department of Chemistry and Biochemistry, Samford University, Birmingham, AL 35229; email:
[email protected]
ABSTRACT
Fungal laccases are a popular objects of exploration due to their involvement in the transformation of
polyphenolic compounds including ethinyl estradiol, better known as birth control. The success of a
laccase in degrading these endocrine disrupting compounds is strongly based upon its electrochemical
potential or E0. Active site consists of 4 Cu2+ centers: the T1 site which is the primary electron acceptor
from reducing substrate. This site has a single Cu2+ center coordinated with two His-nitrogens and one
Cys-thiolate, the T2 which has a single Cu2+ center coordinated to two His-nitrogens and one hydroxide,
and the T3 Binuclear Cu2+ center bridged by one O atom where three His-nitrogens are coordinated at
each Cu2+ site. In this project, the following white rot fungi were studied in order to estimate their
reactivity: Trametes versicolor (Turkey Tail) and Agaricus bisporus (Portobello). The activity of these
“yellow” fungal laccases was attempted to be estimated by measuring the change in absorbance of each
of the following redox indicator solution pairs: Fe(CN)63-/ Fe(CN)64-, Fe(bpy)23+/Fe(bpy)22+, Fe(bpy)33+/
Fe(bpy)32+, Fe(phen)33+/ Fe(phen)32+.By using the Nernst equation, the absorbance of the redox pairs can
directly tell the electrochemical potential of the laccase. T1 redox potential (vs. NHE) for Trametes
versicolor has previously been characterized (800 mV)
INTRODUCTION
Endocrine-disrupting compounds (EDCs) have been detected in our environments, ecosystems, and
most importantly our wastewater systems.[1] Domestic water supplies as well as industrial wastewater
supplies are both primary sources of contamination.[3EDCs such as , bisphenol A, ethinyl estradiol, and
estradiol can negatively impact humans and wildlife and, therefore, must be removed from public
drinking water supplies.[2] Many EDCs alter the overall mechanism of estrogen receptors (ERs) in
humans and wildlife.[3] ] Several methods have been used to assist in their degradation, such as
ultraviolet radiation and advanced oxidation treatment. However, these methods either produce human
hazard or harmful byproducts over time.[2] Enzymatic systems, such as the utilization of laccase, is an
eco-friendly and promising approach to degrade the phenolic structures which contribute heavily to the
malignance of EDCs.[4] Laccase is the most efficient method of enzymatic systems because no
additional/expensive cosubstrate or cofactor is required apart from oxygen. [5] Laccases act as a catalyst
in the oxidation of lignolytic structures by reducing oxygen to water.[3] The redox potential of the
laccase depends heavily upon the amount of activity at the T1 active site of the laccase which is key in
the degredation of phenolic structures. [3] In this project, we aim to measure the redox potential of the
“blue” T1 copper site using a series of redox indicators through the laccases’ change in absorbance. The
redox potential of the laccase will give us a rather accurate indication of how well the laccase will
degrade polyphenolic structures like EDCs.
You need to set it up in the intro why you tried to look at blue laccases, why it didn’t work, what are the
other laccases out there, and how/why you are using blue laccases as the model. Otherwise, it makes
no sense why all the intro is about blue laccases, but the results and discussion are all about yellow
laccases there is not agreement between the
Structure of the Active Site and Enzymatic Mechanism
All laccases consist of a well-conserved active site consisting of four copper centers (Figure 1): (1) T1,
which contains a single Cu2+ ion to which two His-N residues and one Cys-thiolate are coordinated
equatorially and is the primary electron acceptor within the active site; (2) T2, which contains a single
Cu2+ ion to which two His-N’s and one O (as hydroxide) are coordinated; and (3) T3, which contains a
binuclear Cu2+ center bridged by one oxygen atom and to which three His-N residues are coordinated to
each copper ion.[3] Though both the T1 and T2 Cu sites are paramagnetic, only the former exhibits
significant absorption in the UV-visible region (a610nm  5000 M1 cm1); thus, the T1 site is sometimes
called the “blue Cu site.” In contrast, the T3 binuclear site is not paramagnetic and only weakly absorbs
in the near UV (a330nm  2800 M1 cm1).[3]
As oxidases, laccases catalyze four separate one-electron oxidations of a reducing compound; the
electrons generated from these oxidations are then used to reduce dissolved dioxygen gas to two water
molecules. Thus, the mechanism for laccase catalysis involves two substrates: a primary reducing
substrate, which binds at the T1 site and is the source of electrons (and which, in turn, becomes
oxidized). Dioxygen gas, which binds at the T2/T3 trinuclear site where it undergoes reduction to form
water (Figure 1). For reducing substrates such as phenol, the oxidation mechanism requires four
substrate molecules to complete the catalysis, resulting in reactive phenoxy radicals, which can
subsequently undergo radical polymerization:
OH
O
lacc


4H+(aq) +
4e
(1)
+
4e
4H2O(l)
(2)
4
4
O2(aq)
+
+
4H+(aq)

In short, intermolecular electron transfer resulting from the four one-electron oxidations between the
substrate and T1 site is thought to initially lead to a fully reduced form of the enzyme (i.e., one in which
all four Cu centers are reduced to Cu+ ions), the rate of which is not kinetically limiting. The overall
catalytic rate is considered to be largely controlled by the diffusion of dissolved O2, which binds at the
T2/T3 site and leads to a peroxide (O22) intermediate following electron transfer from the T3 Cu+
centers.[3] Subsequent electron transfer from the T1 and T2 sites further reduces the bound peroxide,
resulting in bond cleavage and generation of water molecules.[3]
The ability of laccases to effect the oxidation of phenolic (and non-phenolic) compounds is largely
dictated by the solution redox potentials of the various Cu centers. Given its primary role in the
mechanism described above, the redox potential of the T1 binding site is of particular importance.
Measured T1 redox potentials for laccases from various fungal sources extend over a large range of
values (400-800 mV vs. SHE), where those on the upper end typically reflect greater enzymatic
activity.[3,4] It is well-known that the difference in redox potential of the T1 site for various laccases
and a given reducing substrate is directly proportional to changes in the measured log(kcat/Km), where
kcat is the catalytic rate constant and Km is the Michaelis constant.[5]
METHODOLOGY
This projects methods were based upon the methods decribed in Solomon et al where a series of fungal
laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora hermophila, Scytalidium thermophilum)
and one bilirubin oxidase (Myrothecium verrucaria) were obtained and purified to determine their redox
potentials. These laccases were purified via ion-exhange column and their standard electrochemical
potentials were determined via Cyclovoltametry. Since the duration of the REU program was only 9
weeks long, we decided it would not be very time-efficient to purify the laccases which were harvested.
We then purchased standard laccases from Sigma-Aldrich in order to conserve time.
A glove bag was purchased in order to carry –out the experiment in the absence of oxygen. The glove
bag was to be purged using Argon gas in order to prevent the laccase mechanism from going to
completion. In order for us to examine the activity of the laccase, we needed to stop the mechanism
after the T-sites of the laccase had been reduced in order to fully measure the activity of the laccase.
In this project, we hoped to measure the T1 redox potentials of different fungal laccases using
spectroscopic absorption exhibited by the Cu site.[6] However, due to time restrictions as well as
solubility issues presented by various redox buffers we were not able to reach this fundamental stage of
the project. To date, various redox buffer pairs (Fe(CN)63-/Fe(CN)64-, Fe(bipyridyl)2Cl3/Fe(bipyridyl)2Cl2,
Fe(II)/Fe(III) ortho-phenanthroline(in 1:1, 1:2, 1:3 ratios)) have been considered to be used in
combination with the laccase, but due to persistent problems with solubility were discarded. Many
methods were utilized to try and obtain solubility. The Iron(III)bipyridyl solution was completely
transparent after we made it up in only acidic solution. However, after we diluted the solution with 0.1
M phosphate buffer pH 6 and let it sit overnight it became cloudy and displayed a dramatic color change
from a bright orange to a light pink. We concluded that this may be an issue with the phosphate reacting
with the iron in solution, due to the phosphate being so highly charged. So we changed our buffer to
acetate buffer 0.01 M with pH 5. The solution was then made in basic medium to see if pH was a major
factor affecting the solubility. A 0.2mM Fe(III)Bipyridyl3 solution was made in a pH 9 solution then
diluted in acetate pH 5 buffer. This change still did not yield transparency in the Iron-bipyridyl solutions.
A new complex was then researched and purchased, Tris(2,2 bipyridine) Iron(II) hexafluoro phosphate in
a powder form from Sigma-Aldrich. A 0.5 mM solution was made of this new complex, which was
diluted with acetate buffer. A UV spectrum was taken of this new solution (Figure 2).
The Tris(2,2 bipyridine) redox couple was selected as the best possible redox couple based on
the couples’ high redox potential (refer to table 1) and Tris(2,2 bipyridine) Iron(II) hexafluoro
phosphate’s promising UV-Vis spectrum (see figure below). The Tris(2,2 bipyridine) Iron III hexafluoro
phosphate compound was researched, but it was unavailable to be commercially purchased. However,
we utilized a new boiling method of creating the compound using a boiling flask and a condenser
column.
Trametes versicolor solutions (0.5 µM, 2nd concentration, 3rd concentration) were made to investigate
scattering in UV scans. Potential scattering factors included size of the laccase enzyme or concentration
of the solution. I executed quantitative experiment by taking a 1 mL portion of the original 25mL TV
solution and diluting it to 10 mLs with 9 mLs of phosphate buffer. A UV was then run on the new 10 mL
TV solution and the scan produced a curve similar to the 25 mL scan. I then diluted the solution again by
taking a 1 mL portion of TV out of the 10 mL I recently made and diluting it with 9 Ml of phosphate
buffer. Another UV scan was ran on that solution and it produced a curve identical to the last two. We
came to the conclusion that the scattering effect is not quantitative, it is qualitative. It is an effect
associated with the size of the enzyme itself in solution. I also found a paper which reports a major
increase in UV scattering with laccase.[14] An E˚ value of 800 mV was discovered for the Trametes
Versicolor laccase.[14]
RESULTS
The final results of this project have been delayed due to many obstacles in the lab, such as solubility
and the misinformation about the laccase. We encountered a problem with the standard laccases which
were ordered from Sigma-Aldrich where we initially thought that the standards were “blue” laccases,
however, it was later discovered that the laccases were “yellow” laccases and therefore our
methodology approach to measuring the potential of our laccases has to be altered to where we
measured the change in absorbance of a redox pair solution used in combination with the laccases in
+
order to measure the absorbance. One e- couple must match that for the T1 site in laccase (Cu2+
).
One form of the redox pair is dictated by the extent to which its E° matches that expected for a given
laccase. E°’s probably should be within 100-200 mV of each other due to requirements dictated both by
the Nernst equation and by ability to detect absorption changes. We were first convinced that the
Trametes versicolor and the other laccases were “blue” laccases, which absorb in the 610 nm region
when we received them. However, we discovered that yellow as well as white laccases exist and that
our fungi samples were indeed yellow laccases which do not absorb in the 610 nm region.[16] Since this
discovery we have been recently trying to find a redox mediator pair, as described above, which absorbs
in the same region as the laccase. Recently, a new method has been executed to gain solubility of the
iron bipyridyl solutions using a boiling flask and condenser column set up. The solutions are heated to
boiling with 0.2 mM iron 2 or 3 solution and 2.5 mL of 0.1 M HCl inside and the bipyridyl is added
gradually to prevent it from polymerizing with the iron. The polymerization inhibits the solubility of the
solution as it presents a cloudy appearance as well as a precipitate. We are currently executing this
method with the bis-bipyridine and iron2 and 3 in 0.2mM concentrations at a 25 mL volume.
REFERENCES
[1]
B. Viswanath, B. Rajesh, A. Janardhan, A.P. Kumar, and G. Narasimha, “Fungal
Laccases and Their Applications in Bioremediation,” Enzy. Res. 2014, Article ID
163242, 1-21.
[2]
Degradation of Endocrine Disrupting Chemicals Bisphenol A, Ethinyl Estradiol, and
Estradiol during UV Photolysis and Advanced Oxidation Processes, Erik J. Rosenfeldt
and and Karl G. Linden*Environmental Science & Technology 2004 38 (20), 5476-5483.
[3]
F. Xu, W. Shin, S.H. Brown, J.A. Wahleithner, U.M. Sundaram, and E.I. Solomon, “A
Study of a Series of Recombinant Fungal Laccases and Bilirubin Oxidase that Exhibit
Significant Differences in Redox Potential, Substrate Specificity, and Stability,”
Biochim. Biophys. Acta 1996, 1292, 303-311.
[4]
Gurpreet Singh Dhillon, Surinder Kaur, Satinder Kaur Brar, In-vitro decolorization of
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grown on brewer's spent grain, International Biodeterioration & Biodegradation, Volume
72, August 2012, Pages 67-75, ISSN 0964-8305.
[5]
Gemma Macellaro, Cinzia Pezzella, Paola Cicatiello, Giovanni Sannia, and Alessandra
Piscitelli, “Fungal Laccases Degradation of Endocrine Disrupting Compounds,” BioMed
Research International, vol. 2014, Article ID 614038, 8 pages, 2014.
doi:10.1155/2014/614038.
[6]
D.W.S. Wong, “Structure and Action Mechanism of Ligninolytic Enzymes,” Appl. Biochem.
Biotechnol. 2009, 157, 174-209.'
[7]
F. Xu, J.J. Kulys, K. Duke, K. Li, K. Krikstopaitis, H.-J. W. Deussen, E. Abbate, V.
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[8]
Jung, Jiyoung, and Louise Wicker. "Laccase mediated conjugation of sugar beet pectin
and the effect on emulsion stability." Food Hydrocolloids 28, no. 1 (2012): 168-173.
[10]
Crecchio, C., Ruggiero, P. and Pizzigallo, M. D. R. (1995), Polyphenoloxidases
immobilized in organic gels: Properties and applications in the detoxification of aromatic
compounds. Biotechnol. Bioeng., 48: 585–591. doi: 10.1002/bit.260480605
[11]
F. Xu, “Oxidation of Phenols, Anilines, and Benzenthiols by Fungal Laccases: Correlation Between
Activity and Redox Potentials as Well as Halide Inhibition,” Biochemistry 1996, 35, 7608-7614.
[12]
K. Piontek, M. Antorini, and T. Choinowski, “Crystal Structure of a Laccase from the Fungus
Trametes versicolor at 1.90-Å Resolution Containing a Full Complement of Coppers,” J. Biol. Chem. 2002,
277, 37663-37669.
[13]
J. Cavallazzi, C.M. Kasuya, and M.A. Soares, “Screening of Inducers for Laccase Production by
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[14]
Juan Godoy-Navajas, María Paz Aguilar-Caballos, Agustina Gómez-Hens, “Automatic
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[15]
Feng Xu, Woonsup Shin, Stephen H. Brown, Jill A. Wahleithner, Uma M. Sundaram, Edward I.
Solomon, A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant
differences in redox potential, substrate specificity, and stability, Biochimica et Biophysica Acta (BBA) Protein Structure and Molecular Enzymology, Volume 1292, Issue 2, 8 February 1996, Pages 303-311,
ISSN 0167-4838.
[16]
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Table 1: E0 values for different redox pair indicators [3,16]
Redox Pair
Eo Value
Fe(CN)63-/ Fe(CN)64
0.36 V
Fe(bpy)23+/ Fe(bpy)22+
0.80 V
Fe(bpy)33+/ Fe(bpy)32+
1.30 V
Fe(phen)33+/ Fe(phen)32+
1.10 V
Figure 1. Generalized electrochemical mechanism associated with the catalytic oxidation of reducing
substrates by laccases.[3]
Figure 2: In this spectrum taken via V-Visible Spectrophotometer, a strong peak was seen around the
500-520 nm region which indicates that this buffer solution could be used in combination with the
laccase.
Figure 3: UV-Vis scan of the 0.5 µM Trametes versicolor solution. Even when the solution was diluted 10fold twice, the spectrum produced was identical to the original solution. Even when the solution was
diluted 10-fold twice, the spectrum produced was identical to the original