RSU Cilvēka fizioloģijas un bioķīmijas katedra. Āris Kaksis , 2014
"Biologic Oxidation and Reduction (Red–Ox) reactions" Pētijums Medicīniskā ķīnija
http://aris.gusc.lv/Ox–RedProcesiLd13.doc Uzdevums studijām Medicīniskā ķīmija.
To determine the value of Ox-Red potential E depending from ratio between concentration the oxidizing form
and reducing form [Ox]/[Red] as well from dilution degree of Ox-Red system.
Into three bulbs you have to pour out 10 mL (V3) 2 M potassium chloride solution and to add the following
amount of oxidizing agent (V1) and reducing agent (V2) (Which one are shown from teacher according
description below). To measure the Ox-Red potential E of prepared Ox-Red system.
Dilution two times of prepared Ox-Red system you have to perform by adding of distilled water H2O2 in equal
amounts V1+V2+V3 = VH O = 30 mL , that means dilution (decreasing) of initial solution concentrations two
2
times •2 . To measure the Ox-Red potential E' of diluted Ox-Red system.
Practical preparation and observation of the Potential for Oxidizing-Reducing equilibrium
 [[Fe (CN ) 6 ]3  ] 
,
[Fe+II(CN)6]4- Nernst's Potential is E=Eo+P•log 
 [[Fe (CN ) ]4  ] 
6


ln( 10)  R  T 2.3...  8.3144  298.15
where Eo = 0.355V and P =
=
= 0.0591 V.
Fn
96485  1
Is known the standard potential Eo = 0.355 V and constant P = 0.0591 V . For preparing solution of
Red–Ox equilibrium mixture you have to take: volume V3=10 mL of potassium chloride solution KCl and also
ready solutions of oxidizing (1) [Fe(CN)6 ]3- and reducing (2) [Fe(CN)6 ]4- agents with certain concentrations
C1 = C2 = 0.01 M in water by using tasked volumes respectively V1 =10 mL and V2 =10 mL for each student
specially from below table. You can certainly use given formulas for calculation of prepared solution
concentrations for each other Red–Ox agent:
C1  V1
C2  V 2
[[Fe+III(CN)6]3-]=
;[[Fe+II(CN)6]4-]=
V1  V 2  V 3
V1  V 2  V 3
Prepared solution has total volume V1+V2+V3=30mL.
Measure the EMF (Electric Motion Forces) potential EMF as difference between Red–Ox(Pt) electrode E
and silver/silver chloride Ag/AgCl, KCl potassium chloride saturated solution reference electrode EAg/AgCl
On hydrogen standard electrode potential scale the value of reference electrode is experimentally detected
known as EAg/AgCl=E-EMF=0.355V–EMF=EAg/AgCl=....V expressing from E=EMF+EAg/AgCl=0.355V.
Let us use given condition for calculation of Red–Ox potential E Nernst’s equation the investigated equilibrium
on platinum electrode (Pt) as E=Eo+P•log(1) ; E=Eo+P•0 and E = Eo = 0.355V .
[Fe+III(CN)6]3-+e-
EMF = E - EAg/AgCl. So well experimentally measured should be E = EMF + EAg/AgCl = 0.355 V
observation
1
2
3
4
5
6
7
8
9
V1, mL
V2, mL
20
19
18
15
10
5
2
1
*0.00001
*0.00001
1
2
5
10
15
18
19
20
C[Fe+III(CN)6]3-,
M = mol/L
6.67E-03
6.33E-03
6.00E-03
5.00E-03
3.33E-03
1.67E-03
6.67E-04
3.33E-04
3.33E-09
E, V
0.727
0.431
0.411
0.383
0.355
0.327
0.299
0.279
-0.017
C[Fe+II(CN)6]4-,
M = mol/L
3.33E-09
3.33E-04
6.67E-04
1.67E-03
3.33E-03
5.00E-03
6.00E-03
6.33E-03
6.67E-03
*level of natural admixture
compounds
After measuring E=0.355 V for
Red–Ox system Nr.5 add to
prepared solution having
V1+V2+V3=30mL total
volume 10 mL of oxidizing (1)
[Fe(CN)6 ]3- agent and EMF1
measure experimentally for got
EAg/AgCl=....V and and explain
research results for calculated
E1= EMF1 + EAg/AgCl
 [[Fe (CN ) 6 ]3  ] 
C1  2V1
C2  V 2
 ; [Fe+III]=
E=0.355V+0.0591•log 
; [Fe+II]=
 [[Fe (CN ) ]4  ] 
2  V1  V 2  V 3
2  V1  V 2  V 3
6


.
E1=0.355+0.0591•log(0.01*20/40/0.01/10*40)=0.355+0.0591*log(2)=0.355+0.0591*0.301=0.373 V
Experimental EMF1 = E1 - EAg/AgCl. So well experimentally measured should be E1 = EMF1 + EAg/AgCl=…..V
1
 [[Fe (CN ) 6 ]3  ] 
,
[Fe+II(CN)6]4- Nernst's Potential is E=Eo+P•log 
 [[Fe (CN ) ]4  ] 
6


Graphical image for given Red–Ox System below has a mathematical base in the form of Nernst's equation.
 [[Fe (CN ) 6 ]3  ] 
 Red–Ox System middle point
E= 0.355V +0.0591•log 
 [[Fe (CN ) ]4  ] 
6


at conditions
 [[Fe (CN ) 6 ]3  ] 

 = 1 as
 [[Fe (CN ) ]4  ] 
6


+III
[[Fe (CN)6]]=[[Fe+II(CN)6]]
make over inflection point in
Red–Ox potential dependence
on component concentration
[[Fe+III(CN)6]3-] as log(1)=0.
What we can observe in
experimental research of
Red–Ox System (half reaction).
[Fe+III(CN)6]3-+e-
Conclusions and
Explaining Red–Ox
System Research
How value of potential E
depends on:
341) concentration of oxidizing form (1) [Fe(CN)6 ] and reducing (2) [Fe(CN)6 ] form;
2) the ratio of Red–Ox System oxidizing/reducing form concentrations in middle point log(1) [Ox]=[Red].
3) What about research for Red–Ox system potential E1 after decreasing ratio in two times log(1/2)?
4) Why it would differs from theoretical point of view in experimental research?
What Your experimental research can indicate about oxygen O2 presence in air to be in contact with solution?
5) What and how much transfer the reducing agent in Red–Ox reactions?
6) Which agent is electron acceptor in Red–Ox reactions?
At T=310 K (37° C)
Figure 3.NAD and NADP NAD+ +H- (2e-+H+)
NADH; Eo=-0.059V standard potencial
H
O
O
(a) oxidized NAD+ +2e-+ H3O+↓(H-↓;+H2O)
NADH+H2O reduced form product
(a) Nicotin-amide adenine di-nucleotide (NAD+) and its
Hydrogen
Transfer
H
↓A
His51
H
H
H
H
H + :O:
H2O + H ↓side A phosphorylated analog NADP+ undergoes reduction to
C C O H - H H O ..
2+
H
H H
O
NADH and NADPH, accepting a hydride :H- ion (two
H H Zn
N
H
C
electrons 2e- and one proton H+) from an oxidizable
+
H
Nicotin-amide
N
:O
N
O
substrate. The hydride :H- ion is added to either the front (the
N
H
P O
Adenine
Ribose
H
H
B side
or A side) or the back (the B side) of the planar nicotin-amide
O
N
ring (seeTable2)
O
O
↓ B side↓ H + H2O
P O
H H N
N
(a) oxidized NAD++ 2e-+H3O+
NADH+ H2O reduced
H
O
H
O
N
O
N
}
D-Ribose
H
O
O H
↑A=log(Io/I)
NAD+
C
H
N
for NADP+ ribose C2’-OH hydroxyl in NADP+ is esterified
with phosphate HO-PO32- as ribose 2’C-O-PO32-
N
H
Ribose
Absorbance measured A=a•C•l proportional to NADH concentration C into solution
Figure 3. (b) The UV absorption spectra of NAD+ and NADH.
Oxidized
Reduction of the nicotin-amide ring produces a new, broad
absorption band with a maximum at 340 nm. The production of
NADH during an enzyme-catalyzed reaction can be conveniently
Reduced
followed by observing the appearance of the absorbance at 340 nm;
extinction coefficient a= 6 200 M-1•cm-1 molar absorbance a=A/C/l
in Beer-Buger-Lambert’s law A=a•C•l shows good sensitivity.
NADH
220 240 260 280 300 320 340 360 380 (b) Wavelength (nm)
2
—→
Red–Ox System tables http://aris.gusc.lv/BioThermodynamics/OxRedBiologicalW.doc
Half-reaction - OxRed systems
Data source
Eo (V) EM(V) E° (V) E°H2O(V) E°37(V)
1.7356 1.6734 1.776
1.9821
1.9742
H2O2+2 H3O++ 2 e- = 4 H2O
Suchotina
+
0.305
0.1806
1.2764
1.48246
1.4251
O2 +2 H3O + e = H2O2+ 2 H2O
David Harris
0.813 0.751 1.2288
1.38334
1.3732
O2g+4 H3O++ 4 e-= 6 H2O
Suchotina
+
0.2889
0.1957
0.9275
1.13355
1.1291
NO3 +3H3O +2e =HNO2+4H2O
University Alberta
+
0.3913 0.3291 0.8351
0.98967 0.95138
NO3 + 2 H3O +2e = NO2 + 3 H2O
David Harris
+
0.2336
0.1714
0.6994
0.80243 0.79365
p-quinone+2H3O +2e =Hydroquinone+2H2O
+
0.2336 0.1715 0.6945
0.7975
0.7937
O2aq+2H3O +2e =H2O2aqua+2H2O
University Alberta
0.783 0.783 0.7690
0.7690
0.7830
Fe3+ + e- = Fe2+
University Alberta
+
0.0197
0.0819
0.4591
0.56215
0.5404
Ubiquinone+2H3O +2e =Ubiquinol+2H2O
2+
20.0332 0.0953 0.4451
0.54815 0.52695
Fumarate +2H3O +2e =Succinate +2H2O
+
-0.0774 -0.1395 0.3991
0.50215 0.48273
CrotonylCoA+2H3O +2e =ButyrylCoA+2H2O
-.0862 -.1483 0.3900
0.4930 0.47395
C6H6O6+2H3O+ +2e-=AscorbicAcid+2H2O
DC.Harris
+
-0.111
-0.171
0.324
0.42715
0.42715
Glyoxylate+2H3O +2e =glycolate+2H2O
D.C.Harris 25°C
3+
2+
0.3509 0.3509 0.3650
0.3650
0.3509
Cytochrome F Fe + e = Fe
David Harris
0.3258 0.3258 0.3557
0.3557
0.3258
[FeIII(CN)6]3-+ e-= [FeII(CN)6]4University Alberta
2+
2-0.2225 -0.2847 0.2481
0.35115 0.33757
Oxalo-acetate +2H3O +2e =Malate +2H2O
3+
2+
0.3365
0.3365
0.3500
0.3500
0.3365
Cytochrome a3 Fe + e = Fe
+
-0.2408 -0.3030 0.2291
0.33215
0.3193
Pyruvate +2H3O +2e =lactate +2H2O
0.29815 0.28662
FADfree+2H3O+ +2e-=FADH2 +2H2O
* -0.2735 -0.3356 0.1951
0.2930 0.28169
CH3CHO+2H3O++2e-=CH3CH2OH+2H2O
Kortly Shucha -0.2784 -0.3406 0.1900
0.2788 0.2788 0.290
0.290
0.2788
Cytochrome a Fe3+ +e-= Fe2+
+
-0.2841
-0.3462
0.1841
0.28715
0.27604
GlutaS-Sthione+2H3O +2e =2GlutathSH+2H2O
-0.2859 -0.3480 0.1739
0.27693 0.27424
Srhb+2H3O+ +2e-=HSH+2H2O
University Alberta
0.2442 0.2442 0.254
0.254
0.2442
Cytochrome c Fe3+ + e- = Fe2+
+
-0.3417 -0.4039 0.1241
0.22715 0.21837
LipoicAcidS-S+2H3O +2e =LipSHSH+2H2O
0.2115 0..2115 0.220
0.220
0.2115
Cytochrome c1 Fe3+ + e-- = Fe2+
+
-0.3956 -0.4577 0.0681
0.17115 0.16453
AcetoAcetate +2H3O +2e = -OHButyrate +2H2O
-0.4283 -0.4904 0.0341
0.13715 0.13185
Ketoglutarate2-+CO2+2H3O++2e-=isocitrate2-+2H2O
+
-0.4611
-0.5232
0.000
0.10303 0.09904
H3O + e =H(Pt) + H2O
3+
2+
0.074 0.074 0.077
0.077
0.074
Cytochrome b Fe + e = Fe
+
-0.5784
-0.6407
-0.118
0.03654
0.03513
CH3COOH+2H3O +2e =CH3CHO+3H2O
Suchotina
-0.5873 -0.6496 -0.1314
-0.0284 -0.0273
13PGlycerate4-+ 2H3O++2e-=Glycaldeh3-P2-+2H2O+Pi2+
+
-0.3429
-0.3740
-0.117
-0.0654 -0.0629
NADP +H3O +2e =NADPH+ H2O
+
+
-0.3391 -0.3702 -0.113
-0.0614 -0.0590
NAD +H3O +2 e =NADH + H2O
David Harris
-0.2355 -0.2355 -0.245
-0.245 -0.2355
O2g + e- = O-2aq
Suchotina
3+
2+
-0.415
-0.415
-0.432
-0.432
-0.415
Ferredoxin Fe + e = ferredoxin Fe
+
-.9975 -1.060 -0.5427
-0.4397 -0.4373
2C3H4O3 + 4H3O + 4e = C6H12O6 + 4H2O
Stryer
-0.5938
-0.6559
-0.828
-0.9311 -0.8951
H2O + e = H(Pt) + OH
Suchotina
Table 1. Standard Reduction Potentials Eo and EM of Some Biologically Important Half-Reactions, at 37 °C
for pH=7.36 and 8.37 (in mitochondria), E° at standard conditions 298.16 K, pH=0 for H+/ H reference
electrode E°=0.00 V, E°H2O corrected to water concentration [H2O] = 997.07/18.0153 = 55.3457 M from
equations where involved, and E°37. at body temperature conditions 310.16 K (37 °C) calculated from E°H2O
Data mostly from: 1. Loach, P.A. (I 976) In Handbook of Biochemistry and Molecular Biology,
2. 3rd ed-n (Fasman, G.D. ed.), Physical and Chemical Data, Vol. 1, pp. 122-130 e, CRC Press,
3. A.M.Suchotina, Handbook of Electro-Chemistry, Petersburg ,1981."Chimia"©
4. S.Kortly and L.Shucha. Handbook of chemical equilibria in analytical chemistry. 1985.Ellis Horwood Ltd.©
5. University Alberta Data Tables Molar Thermodynamic Properties of Pure Substances 1997.,
http://www.vhem.ualberta.ca/courses/plambeck/p101/p00403.htm
6. Boca Raton, FL. ''This is the value for free FAD;
FAD bound to a specific flavo-protein (for example succinate dehydrogenase) has a different E°
7. David A. Harris, "Bio-energetic at a Glance". Blackwell Science Ltd ©, 1995, p.116.
8. Daniel C.Harris, "Quantitative chemical analysis". W.H.Freeman and Company ©, 5th ed.1999, p545
3
ln(10) • R• T  [Ox] 
•lg 
 , where E° - standard potential of given OxRed system measured at
F•n
[Re d]
conditions when E = E° (as [Ox]=[Red]); natural logarithm of number 10 - ln(10) = 2.302585093 ;
universal gas constant R=8.3144 J/mol/K; absolute thermodynamics temperature T=273.16°+25°(C)=298.16 K
at standard temperature conditions measured : as Kelvin scale value 273.16 K at zero 0° C point plus on
Celsius scale measured 25°C but human body temperature 37°C that will be higher T=273.16+ 37°C=310.16 K
non-standard conditions; Faraday's constant - F = 96 485 C (coulomb) 1 mol of electrons electric charge in C;
At 298 K (25 °C) and at 310.16 K (37 °C), this expression reduces to respectively following expressions:
E = E°+
E = E°+
0. 05916V  [Ox] 
0. 06154V  [Ox] 
•lg 
•lg 
 ; E = E°+

n
n
[Re d]
[Re d]
Many half-reactions of interest to biochemists involve protons H+ or thermodynamically corrected reality
hydronium ion H3O+. As in the definition of G°. biochemists define the standard state for oxidationReduction reactions as pH 7.36 and express the standard reduction potential as Eo, the standard reduction
potential at pH 7.36. The standard reduction potentials given in Table 1 and used throughout this book are
values for Eo and are therefore only valid for systems at neutral pH. Each value represents the potential
difference when the conjugate Red-Ox pair, at equal concentrations [Ox] = [Red] and pH = 7.36, is connected
with the standard (pH=0) hydrogen electrode. Notice in Table 1 that when the conjugate pair H+ / H at pH 7
is connected with the standard hydrogen electrode (pH 0), electrons e- tend to flow from the pH 7 cell  to
the standard (pH 0) cell; the measured Eo for the H+ / H pair according (3) is - 0.41412 V
B3 vitamins (nikotinic acid, niacin, nikotinic acid amide); NAD structure: nikotin adenin dinucleotide.

Oxidised Form NAD+ + H- (2e-+H+)
His51
H
H
H H
:O:
+
H
H C C O
- H H O ..
2+ H
H
H H Zn
N
Nicotin-amide
:O
O
O P O
N
+
H
NADH Reduced Form Eo=-0.37V standard potencial T=310 K
H
H
H
C C
H
..
O H :O: H
H H
O
H
Nicotin-amide
..
O
O
O P O
H
niacin
nikotinic acid amide
NAD
Nikotin adenin dinucleotide
N
Two reducing equivalents
hydrogen
atoms 2H carrier
Adenine
Adenine
H
several OxRed enzyme
H
H
H
O
O
N
N
coenzyme B3 vitamin
O
O
O
O
N
O P O
O P O
Reduced form NADH + H+
H H
H H N
N
N
destiny acidity increase.
O
O
N
O N
O
N
N
Ordinary reduced form adding
hydrogen atoms never acified
D-Ribose
water medium.
D-Ribose
H
H
O
O
H O
H O
http://aris.gusc.lv/RedOxLehnigerHSGCO2-7-0512.xls strong reducing agent two electrons&proton (+2e-+H+) carrier
with strong power transferees two electrons and one hydrogen ion as hydride H- ion
A Few Types of Coenzymes and Proteins Serve as Universal Electron Carriers
The multitude of enzymes that catalyze cellular oxidations channel electrons e- from their thousands
≈1000 of different substrates into just a few types of universal electron carriers. The reduction of these
carriers in catabolic processes results in the conservation of free energy released by substrate oxidation.
NAD+, NADP+, FMN, and FAD are water-soluble coenzymes that undergo reversible 
 oxidation and
reduction in many of the electron-transfer e reactions of metabolism. The nucleotides NAD+ and NADP+
move readily from one enzyme to  another; the flavin nucleotides FMN and FAD are usually very tightly
bound to the enzymes, called flavo-proteins, for which they serve as prosthetic groups. Lipid-soluble quinones
such as ubiquinone and plasto-quinone act as electron carriers and proton donors in the non-aqueous
environment of membranes. Iron-sulfur proteins and cytochromes, which have tightly bound prosthetic
groups that undergo reversible 
 oxidation and reduction, also serve as electron e- carriers in many
oxidation-Reduction reactions. Some of these proteins are water-soluble, but others are peripheral or integral
4
}
N
H
}
membrane proteins.
We conclude this chapter by describing some chemical features of nucleotide coenzymes and some of the
enzymes (dehydrogenases and flavo-proteins) that use them. The oxidation- reduction chemistry of
quinones, iron-sulfur proteins, and cytochromes is discussed in Oxidative Phosphorylation and PhotoPhosphorylation.
Flavin Nucleotides Are Tighty Bound in Flavo-proteins
 (semi-quinone)
↓ flavin mono-nucleotide FMN→
O
N
H3 C
H
N
H3 C
N
N
H C H
H C O H
FMN H C O H
H
C
H C
O
O P
O
O P
O
FAD
H
H3 C
Ribose
N
O
H
•
N
N O
isoalloxazine ring
+e-+H+→FADH*(FMNH*)
+e-+H+→FADH2 (FMNH2)
H
H
O
N
N
O
H
H
H3C
N
H3 C
Ribose
N
O
N
N
Adenine
O H
Table 3. Some Enzymes
(Flavo-proteins) That
Employ Flavin Nucleotide
Coenzymes
Figure 4. Structures of oxidized and reduced
FAD and FMN. FMN consists of the structure
above the dashed line shown on the oxidized (FAD)
structure. The flavin nucleotides accept two
hydrogen 2H atoms (two electrons 2e- and two
protons 2H+), both of which appear in the flavin
ring system. When FAD or FMN accepts only one
1 hydrogen H atom, the semi-quinone, a stable free
radical, forms.
H
N
N
O
O
N
O
O H
H
H3 C
+
N O
H
Flavin adenine dinucleotide FA D
Flavin Nucleotides Are Tightl y Bound in
Flavo-proteins
←—————————
Enzyme
Flavin nucleotide
Enzyme
Fatty acyl-CoA dehydrogenase
FAD
Di-hydro-lipoyl dehydrogenase
FAD Glycerol 3-phosphate dehydrogenase
Succinate dehydrogenase
FAD Thio-Redoxin reductase
NADH dehydrogenase Complex1 FMN Glycolate dehydrogenase
(fully reduced)
Flavo-proteins (Table 3) are enzymes that catalyze oxidation-Reduction reactions using either flavin
mono-nucleotide (FMN) or flavin adenine dinucleotide (FAD) as coenzyme (Fig. 4). These coenzymes are
derived from the vitamin riboflavin. The fused ring structure of flavin nucleotides (the isoalloxazine ring)
undergoes reversible reduction, accepting either one 1 or two 2 electrons e- in the form of one 1 or two
hydrogen 2H atoms (each atom an electron e- plus a proton H+) from a reduced substrate. The fully reduced
forms are abbreviated FADH2 and FMNH2. When a fully oxidized flavin nucleotide accepts only one 1
electron e- (one hydrogen H atom), the semi-quinone form of the isoalloxazine ring is produced, abbreviated
FADH* and FMNH*. Because flavo-proteins can participate in either one-1 or two electron 2e- transfers, this
class of proteins is involved in a greater diversity of reactions than the pyridine nucleotide-linked
dehydrogenases.
Like the nicotin-amide coenzymes (Fig. 14-15), the flavin nucleotides undergo a shift in a major
absorption band on reduction. Oxidized flavo-proteins generally have an absorption maximum near 570 nm;
when reduced, the absorption maximum shifts  to about 450 nm. This change can be used to assay reactions
involving a flavo-protein.
The flavin nucleotide in most flavo-proteins is bound rather tightly to the protein, and in some enzymes,
such as succinate dehydrogenase, it is bound covalently. Such tightly bound coenzymes are properly called
prosthetic groups. They do not transfer electrons e- by diffusing from one 1 enzyme to another second 2nd;
rather, they provide a means by which the flavo-protein can temporarily hold electrons e- while it catalyzes
electron e- transfer from a reduced substrate to an electron e- acceptor. One important feature of the flavoproteins is the variability in the standard reduction potential (E°) of the bound flavin nucleotide. Tight
association between the enzyme and prosthetic group confers on the flavin ring a reduction potential E
typical of that particular flavo-protein, sometimes quite different from that of the free flavin nucleotide. FAD
bound to succinate dehydrogenase, for example, has an EM = 0.0953 V compared with -0.3356 V for free
FAD. Flavo-proteins are often very complex: some have, in addition to a flavin nucleotide. tightly bound
inorganic ions (iron Fen+ or molybdenum Mon+ coordinative number N=6 bound to protein molecule as
prosthetic group, for example) capable of participating in electron e- transfer because change of oxidation
number (iron Fe2+, Fe3+, Fe4+ or
molybdenum Mo2+, Mo3+, Mo4+, Mo6+).
5