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Biomacromolecules 4/5

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Colin Campbell

on 17 January 2018

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Transcript of Biomacromolecules 4/5

Protein


RNA


DNA
Not all electrons go into the electron transfer chain
In our cells redox potential is (mostly) controlled by glutathione.
GSSG + 2e + 2H 2GSH
(Hopkins and Dixon, JBC 1922)
Biomacromolecules 4/5
Cellular Electrochemistry

Colin Campbell
colin.campbell@ed.ac.uk
Office 280
Cellular energy production
Watch the video
To understand the energetics of this process we need to revisit some basic electrochemistry
V
To measure thermodynamic driving force for reaction – instead of allowing current to flow measure the driving force with no current

Driving force is Voltage (potential difference, electromotive force, EMF)

Measure using a voltmeter (high impedance – negligible current flow)
Many cell voltages have been measured experimentally

Measured cell voltage will change with concentration of redox active ions and pressure of redox active gases

In order to be able to compare values, we measure under “standard conditions”
Measurement of cell voltage
Standard conditions
All activities = 1 (approximately equal to all soluble species concentration 1M and pressures of all gases at 1atm.
All substances are pure
Inert Pt electrodes
Specified temperature (usually 298K)
Measurement of cell voltage
Cell notation takes the form

LH electrode | LH solution || RH solution | RH electrode

Where | denotes a phase boundary, e.g. solid/liquid
and || denotes the salt bridge. All the external circuitry is assumed.
So for our example, cell is:
Cells and notation
Convention is cell voltage is potential of RH electrode wrt LH electrode


so for our cell, the cell voltage is
Cells and notation

where n is the number of electrons transferred in the half cell reactions. F is Faraday’s constant = 96487 C mol-1
Free energy relationship
Cell reaction
Work out how much energy is generated per mole of oxygen in the mitochondrial electron transfer chain, under standard conditions.
The relationship between electrode potential and concentration is called the Nernst Equation.

For a half cell:
E is the new potential
Eθ is the standard reduction potential
R is the gas constant = 8.3143 JK-1mol-1
T is temp/K
xOx + ne- yRed
Nernst Equation



E for an electron transfer process is a function of the concentrations of the participants and the temperature.

Remember how we defined standard reduction potentials?
The Nernst Equation
The reaction is Cu (aq) + 2e Cu(s)


[Cu] can be ignored in the above equation since as an element in the standard state = 1.



What happens to the potential if [Cu ] is 0.01 M or 100M?
Copper example
What difference does it make to the cell voltage if there’s a 10fold difference in conc?
minus
Produced by subtracting two half cell Nernst equations. So for the Zn/Cu cell that we looked at previously:
Nernst equation for a cell
Cellular redox regulation
nucleus
Mitochondrion
endoplasmic
reticulum

Free Radical Biology & Medicine, Vol. 30, No. 11, pp. 1191–1212, 2001
+ve
Oxidative Stress
Normoxia
Hypoxia
-ve
HS
SH
S
S
Proliferation
Differentiation
Necrosis
Apoptosis
cytoplasm
Redox Potential in cells
NADH, NADPH
Zinc dissolves and copper precipitates

Reaction is spontaneous (-ve ΔdeltaG)
Cu
Cu
Zn
2e-
Zn
Consider the simple redox reaction in solution
(Zn added to CuSO4 solution)

Zn(s) + Cu (aq) Cu(s) + Zn (aq)
Electrochemical cells
Redox control of transcription

Transcription factors
HIF
p53
NRF2
NFkappaB
Epigenetics.


DNA methylation/demethylation (associated with transcriptional silencing




Diseases of ageing
Neurodegeneration
Alzheimer's
Macular Degeneration
Cancer
Inflammation
Heart disease
Amino Acids
Redox Pathway Map
TP53
Structure 18, 571–583, May 12, 2010
Apotosis - why is it important
Development - "sculpting" unneeded cells.
Defence - killing infected cells.
Homeostatis - maintaining the right number of cells.
Cancer prevention - getting rid of cells with DNA damage.
Can we use the same approach to represent energy production in the mitochondrial electron transfer chain?
Cyr et al, ANTIOXIDANTS & REDOX SIGNALING Volume 15, Number 2, 2011
Loenarz, C. & Schofield, C. J. Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends in biochemical sciences 36, 7–18 (2011).
Huang, F. et al. Free radical biology & medicine 46, 1186–96 (2009).
Pathway mapping
"A good sketch is better than a long speech"
The "post-genomic" era is characterised by large amounts of biological data
Biology (like chemistry) is driven by interactions and transformations
Cells are complex systems and difficult to interpret intuitively.
There is a need for a standard "graphical notation" to represent biological networks/systems (imagine chemistry without chemdraw).
Mallikarjun, Free Radical Biology and Medicine, 2012, 53, 280-288
Requirements of the ideal cellular redox monitoring system
Useful for quantitative live-cell, non-invasive monitoring
Subcellular resolution to look at specific organelles
Engineered to monitor across the entire pathophysiological potential range.
Pro
ratiometric
potential for sub-cellular resolution
GFP is well known

Con
Slow to respond to changes in potential
can be improved by conjugating with glutaredoxin(Gutscher Nat. Meth. 2008)
Difficult to engineer to other potential windows
Hanson, JBC, Vol 270, 2004
I often say that when you can measure what you are speaking about and express it in numbers you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meagre and unsatisfactory kind.—
Lord Kelvin in Popular Lectures and Addresses, lecture on Electrical Units of Measurement, 1883
Ochsenkuhn et al, ACS Nano 2009
pH Measurement
HQ-NS
Measurement and quantification
You can find this presentation at
http://goo.gl/VOL5vF
2+
2+
2+
2+
-
-
+
2+
2+
Oxidation state of Cys-62 is important in controlling function.
Thinking of the mitochonrial membrane proton gradient in terms of an electrochemical cell.
How much energy for ATP synthesis (per mole of H at 25 C) is stored in a system where the pH outside the membrane is 6, the pH inside the membrane is 7 and the membrane potential is -100 mV?
+
0
NF B is another important pro-survival
transcription factor
kappa
Phillips, T. (2008) The role of methylation in gene expression. Nature Education 1(1)
http://www.nature.com/nrg/journal/v10/n5/pdf/nrg2540.pdf
oxidation
8oxodG:dC
8oxodG:dA
deoxyguanosine (dG)
8-oxo deoxyguanosine (dG)
"the biology of tumors can no longer be understood simply by enumerating the traits of the cancer cells but instead must encompass the contributions of the ‘‘tumor microenvironment’’ to tumorigenesis"
Hanahan and Weinberg, Cell, 2011, 144, 646
Phosphatidylinositide 3-kinase
PI3K is activated by binding to the receptor
Growth factors bind, change the receptor shape and enable PI3K binding.
Phosphatidylinositol 4,5-bisphosphate
Protein Kinase B
Low Mitochondrial Energy
Demand
High Mitochondrial Energy
Demand
Low NADH turnover
Low oxygen demand
High ROS production
High NADH turnover
High oxygen demand
Low ROS production
10% of lung cancer patients have mutations in NRF2.
a.k.a NRF2
1 in 30,000 have VHL mutations that lead to tumor formation
Biochemistry, 2013, 52, 6412
Metabolic consequences of Hypoxia
phosphatidylinositol 3,4,5 triphosphate
Epigenetic modification is a dynamic process that provides a link between environmental factors and disease.
Histone methylation/demethylation
Regulates recruitment of chromatin modifying enzymes
Ma et al, NQO1 overexpression is associated with poor prognosis in squamous cell carcinoma of the uterine cervix. BMC Cancer 2014, 14:414
Antioxidant response genes
Phosphatase and Tensin homologue
3D Tumor Spheriod
Hypoxia
Metabolism
Drug penetration
Nutrients
Waste products

Outer
Intermediate
Core
untreated
1 Gy
2 Gy
4 Gy
8 Gy
Life is like riding a bicycle.
To keep your balance you must keep moving.

Albert Einstein
Thurlow et al, Analytical Chem, 2016, 88, 2727
http://www.optima-cdt.ac.uk
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