Photobiocathode
Proteins: main properties and their
immobilization on the surfaces
Most enzymes are proteins
Attachment of enzyme molecules to the surface
Chemical adsorption (covalent bond)
enzyme
Physical adsorption (van der Waals, electrostatic, hydrophobic,
DLVO, non-DLVO)
linking molecule
enzyme-
substrate
Au, Si, carbon, polymer
Thiol group (Au-S-(CxHyRz)-HN2-enzyme)
Si-C bond (Si-C- (CxHyRz)-HN2-enzyme)
Enzyme film on electrode surface
Site specific adsorption
Protein immobilization
Physical adsorption
Chemical adsorption
- hydrophobic/hydrophilic interactions
- covalent bonding of the protein
-electrostatic interactions
to the solid phase
- van der Waals interactions
- experimentally complicated
- experimentally simple
- can alter the conformational structure
- often retain the activity of the proteins
- proteins can be easily removed
and active center of the protein (activity)
The DLVO-theory
is named after Derjaguin, Landau, Verwey and Overbeek.
It was developed in the 1940s.
The theory describes the force between surfaces interacting through a liquid medium.
It combines the effects of the van der Waals attraction and the repulsion.
assumes the stability of a dispersion of particles as consequence of the algebraic sum of
potential energies leading to repulsive and attractive (electrostatic, van der Waals) interactions.
DLVO (long range electrostatic vs. short range (1/r6van der Waals interactions)
and non-DLVO (surface texture, nanotopographies, surface chemistry, steric hydration forces)
forces, the dominating influence is specific to the respective biomolecule-surface chemistry
and structure
Proteins
The linear polymers built of monomer units called amino acids.
C – α carbon (chiral)
H
NH3+
C
R
O
COO-
αC - COOH
αC - C
O
H
I
αC-N:
I
H
H
I
[αC-N H]+
I
H
R – side chain
}-
H
NH3+
C
H
COOH
NH3+
R
>pH
Both groups protonated
C
H
COO-
R
neutral pH
Zwitterionic form
NH2
C
COO-
R
<pH
Both groups deprotonated
Isoelectric point (pI) of a protein is the pH at which its net charge is zero. The protein
does not migrate in the electrical field.
All proteins in all species are constructed from the same set of
20 amino acids
α– carbon atom is achiral
Amino acids with aliphatic side chains
The large aliphatic side chains are hydrophobic.
They tend to cluster together rather than contact water. Stabilization by hydrophobic effect.
The side chain is bonded to both the nitrogen and the α-carbon atoms.
Influence protein architecture because its ring structure makes it more conformational
restricted than other amino acids.
Amino acids with aromatic side chains
hydrophobic
Less hydrophobic due to hydroxyl and NH groups.
Indole ring
The aromatic ring contains delocalized π electrons.
Amino acids with aliphatic hydroxyl side chains
The hydroxyl group makes them much more hydrophilic and reactive than ananine and valine
Amino acids with aliphatic thiol side chains
The sulfhydryl group is much more reactive than –OH. Pairs of sulfhydryl groups may
come together to form disulfide bonds, which are particularly important in stabilizing
some proteins.
The basic amino acids
Histidine often found in the active sites
of enzymes, where the imidazole ring can
bind and release protons in the course of
enzymatic reactions.
-very polar side chains, highly hydrophilic
- positively charged at neutral pH
imidazole group
Amino acids with side chain carboxylates and carboxamides
Acidic side chains
Amino acids with side chain carboxylates and carboxamides
Peptide bonds
- quite stable kinetically, resistant to hydrolysis
- planar, six atoms lie in the same plane
C-N bond has double-bond character
-rotation about the bond between
α – carbon and nitrogen
α – carbon and carbonyl carbon
Proteins – polypeptide chains contain between 50 and 2000 amino acids
Peptides – polypeptides made of small number of amino acids
Proteins have multiple levels structure.
Primary structure
order of amino acids in protein
Secondary Structure
Stabilization role of hydrogen bond
CO of carboxyl groups of amino acid are hydrogen bonded to the NH in amino groups.
Alpha Helix
Hydrogen bond between n-CO and (n+4)NH.
Chain tightly coiled
The side chains extend outwards in a helical array.
Each residue is related to the next one by a rise of 1.5A along the helix axis.
100 degrees rotation
3.6 amino acids per turn
α-helix is right-handed (clockwise) and is more stable than left-handed β-helix
75% α helix in feritine
Secondary Structure
Beta Sheet
Chain almost fully extended
Is formed by linking two or more β strands by hydrogen bonds
The distance between adjacent amino acids along a β strand
is approximately 3.5A.
The fatty acid-binding proteins (lipid metabolism) are built
Almost entirely from β sheets.
Loop
Hydrogen bond between
n-CO and (n+3)NH.
Loops lie on the surface of proteins and often participate
in interactions between proteins and other molecules.
Tertiary Structure
Is the full 3-dimensional folded structure of the polypeptide chain.
Hexokinase – metabolic protein
Ferritin – iron storage protein
Quaternary Structure
Is only present if there is more than one polypeptide chain. With multiple polypeptide
chains, quaternary structure is their interconnections and organization.
Hemoglobin – four polypeptides and heme groups
Enzymes
which bind metal ions are known as metallo-enzymes: in these enzymes the metal cofactor is
usually found at the active site of the enzyme, where it may have either a structural or a
catalytic role.
Redox-active Metallo-enzymes
The other common role of metal ions is as redox reagents. Since none of the 20 common
amino acids are able to perform any useful catalytic redox chemistry, it is not surprising that
many redox enzymes employ redox-active metal ions.
P R O T E I N F I L M V O LTA M M E T R Y
A Photobiocathode
Energy flow in nature
ORR
ATP
ADP
Nicotinamide adenine dinucleotide phosphate(NADPH)
Nicotinamide adenine dinucleotide (NADH)
nicotinamide
ribose
ribose
adenine
+2e-+H+
Flavin adenine dinucleotide FAD- redox cofactor
Riboflavin
Adenosine diphosphate
oxygen reduction reaction: ORR
Laccase
1.229 V vs.NHE
0.67 V vs. NHE
Horseradish peroxidase
HRP
1.77 V vs. NHE
Biomimetic approach
organic – inorganic hybrid
to mimic light involved processes
systems based on semiconductor materials
charge transfer semiconductor – enzyme active center ???
Electrochemistry at semiconductor surfaces
material
for photoanode
material
for photocathode
[V]
P H O T O B I O C AT H O D E
Enzyme activation by photo-induced electrons from semiconductor conduction band
Device concept for direct charge transfer
substrates
inorganic
active center
products
enzyme
Enzyme absorption on semiconductor surface without linker molecules maintaining the activity
of enzyme
Substrate
Step Bunched p-Si Surface (SBpSiS):Substrate for enzyme adsorption
Photo-Electro-Nanostructuring in NaOH solution
p-Si(111)
Terrace:
height 1.5-2 nm (majority)
widths 150-200 nm
Less defined structure than for n-type Si
- oxidation at OCP
- synchronization oxidation / etchingTM
AFM
TEM cross section
“In system” surface analysis
Synchrotron Radiation Photoelectron Spectroscopy (SRPS)
low CxHy coverage (<1Å)
after electrochem. conditioning
Bessy
U49/2
K. Skorupska, J. Solid State Electrochem. 13(2009)205
Synchrotron Radiation Photoelectron Spectroscopy (SERPS) analysis
Si 2p
hν=150 eV
λ=2Å
=Si
Si-H
=Si
H
H
H
Electronic situation
valence band onset
=Si
H
OH
suboxides oxide
negative charge at the surface
site-specific adsorption
enzyme molecules at the semiconductor surface:
Reverse Transcriptase RT
+
_
Laccase from Trametes versicolor
Cu atoms
at the laccase
active center
copper-containing oxidase
T1 (Cu1), T3(Cu1, Cu2), T2(Cu4)
Oxygen reduction
60-70 kDa
diameter about 5 nm
α-helices
β-strands
His – Cys -His
Biosensors and bioelectronics 20(2005)2517
T1 center, which is the substrate reaction site and e- acceptor
site
T3 binuclear type
T2 center is labile, and reduction can selectively remove this
copper ion generating the T2 depleted
T2 and T3 centers are close together and form a trinuclear
cluster, which is the site of dioxygen reduction
T1 connected to trinuclear T2, T3 by His-Cys-His tripeptide
Chem. Rev. 96(1996)2563
reaction with
J. Am. Chem. Soc. 124(2002)6180
decays to the
resting oxidized
Experimental protocol for PhotoBioCathode preparation
enzyme
Coating with Nafion inhibiting desorption of enzyme from surface
Without Nafion the photobiocathode is much less efficient
enzyme
Activity of enzyme molecules at the semiconductor surface are preserved due to
presence of biological water
I -static water layer (directly bound (0.4 nm))
II – quasi-free (0.7 nm))
III bulk water – III
Lw the border between the direct and
the quasi free water shells (Lw≈ 0.4 nm)
K. Skorupska et al J. Electroanal. Chem. 662(2011)169
PhotoBioCathode: TM AFM investigations
E- donor substrate binds in a small negatively charged cavity
near T1 Cu site
7Å below the surface of enzyme
indication of side specific adsorption
Laccase - diameter about 5 nm
Tip – diameter ~10nm
no Nafion overcoating
blue for cations
red for anions
polar uncharged (violet)
and hydrophobic (gray)
(Photo)electrochemical analysis of the
semiconductor-enzyme hybrids
I. Laccase – Step Bunched pSi Surface
forward current
0.1 M citric acid and a 0.2 M K2HPO4 pH 5.2.
I-W lamp 50 mW cm-2
0.1 M citric acid and a 0.2 M K2HPO4 pH 5.2.
I-W lamp 50 mW cm-2
Charge transfer via Carbon NanoTubes
p-Si : CNT : laccase
p-Si : CNT
illumination: I-W lamp 60 mW cm-2
Electrolyte: citric acid, K2HPO4, pH=5.2
O2 saturated
55
Electrochemical characterization of photobiocathode for different light energies
0.1 M citric acid and a 0.2 M K2HPO4 pH 5.2.
(Photo)electrochemical analysis of the
semiconductor-enzyme hybrids
II HRP – Step Bunched pSi Surface
HRP (Horseradish peroxidase)
Horseradish peroxidase HRP
heme as a cofactor
porphyrin
Hydrogen peroxide reduction
N2
H2O2
with enzyme
phosphate buffer pH=6.0
I-W lamp 50 mW cm-2
N2
H2O2
enzyme free
phosphate buffer pH=6.0
I-W lamp 50 mW cm-2
HRP
enzyme free
phosphate buffer pH=6.0
I-W lamp 50 mW cm-2
Origin of overpotential
at photobiocathode
To determine flat band potentials Efb
pH = 5.2
1
2 
kT 
=
 E − E fb −

C SC eεε 0 N 
e 
Csc -
capacitance of the space
charge region
εdielectric constant of the
semiconductor
permittivity of free space
εo Ndonor density (hole
acceptor concentration for a
p-type semiconductor)
Eapplied potential
flatband potential
Efb Mott-Schottky plots (1/C2 vs. E)
Efb=-0.42
Efb=-0.23 V
Efb=-0.32 V
The value of the redox
potential of the T1 Cu-site
has been determined using
potentiometric
titrations
with redox mediators for a
great number of different
Lcs and varies between 430
and 780 mV versus NHE
Hydrogenase at p-Si surface
oxygen-tolerant [NiFe]-hydrogenase
In collaboration with:
Johannes Fritsch, Oliver Lenz
Humboldt-Universität zu Berlin Institute of Biology / Microbiology, Berlin, Germany
hν
J. Fritsch, P. Scheerer, S. Frielingsdorf, S. Kroschinsky, B. Friedrich, O. Lenz, C. M. Spahn,, Nature 479 (2011)249
Electrochemical tests
DEMS
Differential electrochemical
mass spectroscopy
(with help of Dr.P. Bogdanoff)
0.1M phosphate buffer
pH=6, saturated with N2
Light 40 mWcm-2
PS I at step bunched Si surface
Biomolecule modification
PSI
J. Am. Chem. Soc., 2008, 130 (20), pp 6308–6309
69
Site specific adsorption by TM AFM
5-7 nm high
70-100 nm width
PS I size detected by electron micrography
E.J. Boekema et al FEBS Lett. 217(1987)283
Reflectometry analysis
carotenoids
chlorophylls
Measurements performed by Dr. M. Lublow
Thank you for your attention