Introduction
Definitions
Types of cytochromes
Absorption spectrum
Cytochrome p450
Cytochrome a
Cytochrome b
Cytochrome c
Cytochrome d
Hydrogen electron transfer chain
Ubiquinone
Iron-sulfur proteins
Flavoproteins
Complex I: NADH-ubiquinone oxidoreductase
Complex II : succinate ubiquinone oxidoreductase
Complex III: ubiquinol-cytochrome c oxidoreductase
Cytochrome c is a mobile carrier of electrons
Complex IV: Cytochrome c Oxidase
Pathway of electron transfer through complex IV Chemiosmotic hypothesis
ATP Synthase
Bibliography
The aim of this article, which is entitled as “cytochromes and their role in hydrogen electron transfer chain”, is primarily to focus on the general classification, properties and some reactions of cytochromes with particular interest to their role as electron carriers in ETC. Cytochromes are a very large family and until now there is no accurate method for their classification. They play a wide variety of functions in the body and without them life would be impossible. The electron transfer chain is a very complex process that takes place in all living organisms (with slight differences). It is a chain that provides the energy required to synthesize the energy carrier ATP. The process is so complex that until now there are only different hypothesis given for its mechanism and many of its aspects are yet unclear.
Hemeproteins that transfer electrons belong to the family of the cytochromes. The name 'cytochrome' was introduced by Keilin in 1925 to describe a group of intracellular hemeproteins that undergo oxidation-reduction and, upon reduction, exhibit intense absorption bands between 510 and 615 nm. As currently used, the name appears to include all intracellular hemeproteins with the exception of hemoglobin, myoglobin, the peroxidases, catalase, tryptophan 2,3-dioxygenase, heme-thiolate proteins (P-450) and the nitrite and sulfite reductases. Consequently, proteins of markedly different function are found in this family. Thus a number of enzymes are also referred to as cytochromes. These include cytochrome-c oxidase (EC 1.9.3.1), L-lactate dehydrogenase (cytochrome) (yeast cytochrome b2, EC 1.1.2.3) and cytochrome P-450 (EC 1.4.14.1).
The term 'heme' is usually understood as any tetrapyrrolic chelate of iron. The terms 'ferroheme' and ‘ferriheme’ still refer to the Fe (II) and Fe (III) oxidation states in heme; however, the Fe (IV) oxidation level of heme iron is found as a catalytic intermediate in some systems. A hemochrome is defined as a low-spin compound of heme in which fifth and sixth coordination places are occupied by strong field ligands regardless of the oxidation state of the iron. Finally, the terms 'hemoprotein' or, preferably, 'hemeprotein' refer to a protein containing a heme as a prosthetic group.
Ferriheme is sometimes referred to as 'hematin', a usage still sanctioned by tradition. However, this term should not be used for the compound crystallized as the chloride or other salt; such compounds are customarily termed 'hemin'.
The traditional definition that the fifth and sixth coordination positions are occupied by nitrogen atoms was obviously too restrictive as borne out by the case of cytochrome c, wherein one of these sites is occupied by the sulfur atom of methionine.
The classical definition of cytochrome is retained: a cytochrome is a hemeprotein whose characteristic mode of action involves transfer of reducing equivalents associated with a reversible change in oxidation state of the prosthetic group. Formally, this redox change involves a single-electron, reversible equilibrium between the Fe (II) and Fe (III) states of the central iron atom.
Four major groups of cytochromes are currently recognized:
Cytochromes a: Cytochromes in which the heme prosthetic group is heme a, i.e. the iron chelate of cytoporphyrin IX.
Cytochromes b: Cytochromes with protoheme [the iron chelate of protoporphyrin IX] as prosthetic group but which lack a covalent bond between the porphyrin and the protein.
Cytochromes c: Cytochromes with covalent thioether linkages between either or both of the vinyl side chains of protoheme side chains and the protein.
Cytochromes d. Cytochromes with a tetrapyrrolic chelate of iron as prosthetic group in which the degree of conjugation of double bonds is less than in porphyrin, e.g. dihydroporphyrin [chlorin; heme d], tetrahydroporphyrin [isobacteriochlorins; heme d1, siroheme]. Heme d has also been known as heme a2.
Cytochromes are classified according to:
1- a-band of their absorption spectra
2- type of heme group attached to the protein
Differences in their absorption band and standard redox potentials is due to heme differences and its environment around the protein.
The a-band of the reduced cytochromes a is at about 603 nm
The a-band of the reduced cytochromes b is at about 562 nm the b-band at 532 nm
nfor
cyt. c the a-band maximum is at 550 nm, the b-band at 520
nm
Certain variant cytochromes remain difficult to classify. Cytochromes 'o', a type of protoheme oxidase found in prokaryotes, and helicorubin, should be listed as b-type cytochromes.
Cytochrome 'P-450'
Cytochrome 'P-450', which originally received its name in a casual manner, has been characterized as a class of proteins with activity as a monooxygenase involved in hydroxylation associated with electron transfer. Its chemical nature as a b-type cytochrome with atypical non-nitrogenous ligands is well established in a number of systems. Since the characteristic mode of action of these enzymes is not electron transfer (some P-450-enzymes probably do not even involve the reversible Fe(II)/Fe(III) equilibrium), but rather oxygen atom transfer, the name 'cytochrome' did not seem appropriate. Based on the fact that a thiolate ligand at the heme is responsible for the unusual spectral and catalytic properties of these hemoproteins the name 'heme-thiolate proteins' is now recommended.
Cytochrome a group
Cytochrome aa3. The protein complex contains two hemes a, a low-spin component called cytochrome a and a high-spin component called cytochrome a3. The a-band of the reduced cytochromes is at about 605 nm. Cytochrome aa3 is membrane bound and catalyses the oxidation of mitochondrial cytochrome c and some related bacterial proteins by O2. In most cytochromes both positions 5 and 6 are filled by amino acid residues and reaction with O2 is prevented. In hemoglobin position 5 is filled by a histidine of the protein and position 6 is free to bind O2. The same is true for this cytochrome, which reacts with molecular oxygen. In E. coli both cyt d and cyt o are terminal oxidases
Cytochrome a1 has been isolated from Nitrobacter agilis. It has an absorption maximum at 587 nm and is not autoxidizable. Autoxidizable proteins with absorption maxima have been reported for Acetobacter pasteurianum and Escherichia coli.
Haem aposition 2 has a 17-carbon side chain,position 4 is vinyl and position 8 has -CHO
Cytochrome b group
Cytochromes b (cyt b) can be defined as electron transfer proteins having one or two haem b groups, noncovalently bound to the protein. The fifth haem iron ligand is always provided by a histidine residue. Cyt b possesses a wide range of properties and function in a large number of different redox processes. Some haemoproteins, such as P450s and nitric oxide synthase (NOS), have been termed `cytochromes b' as well. Although both P450s and NOS can be considered as b type cytochromes, their main function is catalysis. The recommended name for the group of enzymes which includes P450s and NOS is `haem-thiolate proteins'
Cytochrome b, vinyl groups at both 2 and 4 positions
Cytochrome b1 has been detected in Escherichia coli
Cytochrome b2 is present in yeast.
Cytochrome b5 is present in animal microsomes and the cytoplasm of the erythrocyte
Cytochrome c group
|
Formal iron oxidation/spin states |
|
|
Haem b |
FeII (S=0); FeIII (S=1/2) |
Cytochrome c, the smallest of the cytochromes (molecular weight 12,000) is present in eukaryotic mitochondria where it functions as the substrate for the terminal oxidase (EC 1.9.3.1) in oxidative phosphorylation. It is a soluble, low-spin, monohemeprotein with 103-112 residues. Its midpoint redox potential over most of the physiological pH range is about 250 mV. In its reduced form, the a-band maximum is at 550 nm, the b-band at 520 nm and the Soret peak at 415 nm. Both vinyl groups are present in thioether bonds and the heme iron is coordinated by histidine and methionine. It is the prototypic c-type cytochrome.
Cytochrome c1 is the 30 kDa membrane-bound c-type protein of mitochondria with a-band maximum at 553 nm in the reduced form. It is a low-spin monoheme protein with the same axial ligands as cytochrome c. The mid-point potential is about 270 mV. It functions as electron donor to cytochrome c in the mitochondrial and bacterial respiratory chain. The related protein present in the bc complex of green plants is also called cytochrome f.
Cytochromes c (cyt c) can be defined as electron transfer proteins having one or several heme c groups, bound to the protein by one or, more commonly two, thioether bonds involving sulfhydryl groups of cysteine residues. The fifth heme iron ligand is always provided by a histidine residue.
|
Class |
Heme iron coordination |
Axial iron ligands |
|
c1 |
Hexacoordinate |
N S |
Positions 2 and 4 both are -CH (CH3)-S-Protein
Cytochrome d group
Cytochrome d was earlier known as cytochrome/heme a2. It is present in many aerobic bacteria, especially when grown with a limited oxygen supply. Typical species include Escherichia coli and Aerobacter aerogenes. In protein complexes, it typically gives an absorption band at about 636 nm (oxidized) or 628 nm (reduced). It is found associated with other prosthetic groups in multi-subunit complexes
Hydrogen Electron Transport Chain
The electron transfer chain (also called the electron transport chain, ETC, e-train, or simply electron transport), is any series of protein complexes and lipid-soluble messengers that convert the reductive potential of energized electrons into a cross-membrane proton gradient. This proton motive force created by the ETC is used to power membrane transporters and adenosine triphosphate synthesis by ATP synthase. They are used in photophosphorylation and respiration.
In eukaryotes an ETC is found spanning the inner mitochondrial membrane and accepts electrons from electron donors such as NADH or succinate, shuttle these electrons from within the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space, where they ultimately reduce oxygen.
There are five complexes normally associated with the mitochondrial electron transfer chain.
 | Complex I - NADH dehydrogenase, also called NADH coenzyme Q reductase. |
| Complex II - Succinate - coenzyme Q reductase. |
| Complex III - Coenzyme Q - cytochrome c oxidoreductase. |
| Complex IV - Cytochrome c oxidase. |
| Complex V - ATP synthase, also known as the FoF1 particle. |
All of these are proteolipid complexes, with the first four containing either of the flavin, iron-sulfur clusters, copper centers, or heme moieties. Complexes I, III, and IV are proton pumps. Complex II is part of the Krebs cycle and does not pump protons, and Complex V uses the electrochemical potential generated to create ATP. Complex IV is the terminus of the electron transfer chain, where oxygen from the lungs is reduced by electrons and hydrogen protons (provided by NADH and/or FADH2) to make water.
Ubiquinone.
All ETCs contain quinones. Quinones are hydrogen carriers and carry 2H at a time. The structure of the quinone nucleus varies: ubiquinone (respiration) has single aromatic ring whereas menaquinone (anaerobic respiration) has a double ring. Ubiquinone is sometimes called coenzyme Q and its side chain (R-group) is made of 6-10 isoprenoid (C5) units depending on the organism. Mitochondria n = 10.
There is more ubiquinone than all other ETC components together. A small portion of the ubiquinone is tightly bound to ETC proteins and the rest is free to diffuse around in the membrane - the so-called ubiquinone pool.
The R-group = - [CH2-CH=C (CH3)-CH2] n-H|
Iron-Sulfur Proteins (FeS Proteins).
These have iron but no heme group; instead the iron is linked to acid-labile, i.e. inorganic, sulfur. Sometimes are called non-heme-iron (NHI) proteins. The FeS groups are electron carriers. Each FeS group carries only one electron even though it may have 2 or 4 iron atoms. The electron is shared among the irons:
e- + Fe2+ = Fe3+
Several variant structures, the most common are the flat Fe2S2 and the cube shaped Fe4S4 groups. Both are bound to the protein by four cysteine residues.
![[Fe4S4]Cys4 image](cytochromes_files/image017.gif)
![[Fe2S2] image](cytochromes_files/image018.gif)
[Fe4S4](S
Cys)
4 [Fe2S2](SCys)
4
Flavoproteins
These contain the yellow flavin coenzymes FAD & FMN. The NADH-dehydrogenases are flavoproteins. Several other flavoproteins join the ETC as side branches at ubiquinone, thus missing coupling site #1. Examples are succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glycerol-P dehydrogenase
Flavoproteins may also contain FeS groups (e.g., SDH, NADH-DH I). Usually FAD or FMN is not bound covalently, but occasionally FAD may be bonded to a histidine (e.g. in SDH). Flavoprotein dehydrogenases are linked to the ETC and are not reoxidised by molecular oxygen. In contrast, flavoprotein oxidases are not linked to the ETC and may be reoxidized directly by O2 e.g., aldehyde oxidase, glucose oxidase. Facultative anaerobes such as E. coli have few oxidase type flavoproteins.
Complex I: NADH-Ubiquinon Oxidoreductase
Complex I often called NADH dehydrogenase, (oxidizes NADH and transfers e to ubiquinon (coenzyme Q)), is the most complicated and in mammals consists of 40 different polypeptides with a molecular mass of around 1000000(one million) Da. The first step in oxidation of NADH involves transfer of two e and two protons from NADH to FMN (tightly bounded to one polypeptide subunit in complex I) two e and two protons are added across the double bond of FMN. The electrons are subsequently transferred from FMN via a series of FeS centers (both 2Fe2S and 4Fe4S) to ubiquinon. One important role of FMN is to serve as both two e acceptor from NADH and one e donor to FeS centers. (So it can exist as a stable semiquinon. Four protons are also pumped from the matrix side to intermembrane space (the energy produced by oxidation processes pumps the protons little is known of the mechanism involved)
So finally in complex I two e are transferred to ubiquinon coupled with translocation of two protons.
Complex II: succinate-ubiquinon oxidoreductase
It is also known as succinate dehydrogenase, catalyses the oxidation of succinate to fumarate. The complex consists of four subunits. One subunit contains an FAD as cofactor attached to histidin residues and another subunit that contains three FeS centers and two small hydrophobic proteins. During oxidation of succinate to fumarate two e and then two protons are transferred to FAD. The FADH2 thus formed transfers e to ubiquinon via FeS centers in the following two reactions:
Succinate fumarate +2H+ +2e
UQ +2H+ +2e UQH2
(Over all) succinate +UQ fumarate +UQH2
^E=0.029V
Since
not enough energy is liberated in oxidation of succinate and transfer of e to
ubiquinon no free hence no gain in free energy results in no pumping of protons
in complex II.
Complex III: Ubiquinol- Cytochrome c Oxidoreductase (EC 1.10.2.2)
Complex III also called the cytochrome bc1 complex catalyses the transfer of electron from ubiquinol to cytochrome c with the translocation of protons across the inner mitochondrial membrane. This complex consists of 11 subunits in which three of them contain prosthetic groups and are evolved in redox processes 1- cytochrome b 2- cytochrome c1 3- Rieske iron-sulfur protein which contains a 2Fe2S cluster. of this complex.
Cytochrome c is a mobile carrier of electrons
The iron in the porphyry ring of cytochrome c, is coordinated to N histidin and S of methaionin, thus preventing the interaction of iron with O2.
Cyt. c like ubiquinone functions as the mobile carrier in the electron transport chain. The protein is held loosely to the outer face of the inner membrane by electrostatic forces where it binds to cyt. c1 of complex III and accepts e. The reduced cyt. c then moves along the membrane surface where it interact with subunit II of cyt. c oxidase again through electrostatic linkages and donates e to CuA site.
Complex IV: Cytochrome c Oxidase
Complex IV helps in the transfer of e from cyt c to O2 coupled to translocation of protons across the membrane. This multisubunit complex consists of 13 subunits with molecular mass of 200000 Da in which the redox components are cytochrome a a3 and two Cu centers CuA and CuB. Only three subunits in cyt c oxidase are found to be important in e transfer and pumping action (since these three are found only in bacterial membranes in which catalyses the same reaction). These three subunits which are identical to subunits in bacterial cyt c oxidase are encoded in mitochondrial DNA (mtDNA). The remaining subunits which function as regulatory subunits or in assembly of the enzyme are encoded in nuclear DNA.
Subunit I, the largest polypeptide of the complex contains 12 transmembrane helices.
Two heme groups a and a3 are bound to subunit I such that the iron in protoporphyrin ring is coordinated to N of histidine residue. In addition subunit I contains a copper atom (CuB) that with a3 forms a binuclear center involved in transfer of e from heme a to O2. Subunit II has a large domain protruding to the intermembrane space where reduced cyt c binds and contains two atoms of copper bound through sulfhydryl groups to two cysteine moieties (called CuA). Subunit III contains seven transmembrane helices and hasn’t any redox carriers. The role of subunit III is unclear. Subunits II and III are localized on opposite side of subunit I.
Pathway of electron transfer through complex IV
1- Electrons are transferred from reduced cyt. c to CuA site on subunit II
2- then transferred to heme a of subunit I of complex IV (CuA and cyt. c are localized within 1.5A of each other permitting rapid transfer of e to occur)
3- e are then transferred to binocular center consisting of CuB and a3 where final transfer of e to O2 occur
4- initially two e are transferred to an O2 tightly bound to binocular center to form the peroxy derivative of O2 (O22-)
5- two additional e are transferred to binocular center with concomitant uptake of four protons from the matrix(the pathway for proton uptake is unclear but experimental evidence has suggested that charged amino acids located on subunit I are involved in forming a channel for proton movement. Hence for every four proton taken up from matrix two are pumped into intermembrane space )
NB. Each of the intermediate formed in the reduction of O2 remains tightly bound to the binocular center (the partially reduced O2 such as supper oxide, hydrogen peroxide, or hydroxyl radicals are toxic) until water is produced.
Chemiosmotic hypothesis
Mitchell's chemiosmotic theory postulates that the energy from oxidation of components in the respiratory chain is coupled to the translocation of hydrogen ions from inside to the outside of the inner mitochondrial membrane. The electrochemical potential difference resulting from asymmetric distribution of hydrogen ions is used to drive the ATP synthesis mechanism.
ATP Synthase
The ATP synthase or phosphorylating complex consists of two sucomplex, F0 complex that is a disk of C protein subunits. Attached is a gamma subunit in the form of a bent axle. F1(which project into the matrix and which contain the phosphorylation mechanism) consists of three alpha and three beta subunits which are fixed to the membrane and do not rotate. ADP and P are taken up subsequently by the beta subunit to form ATP which is expelled as the rotating gamma subunits squeezes each beta subunit in turn. The gamma subunits rotate due to protons passing through the C unit. Thus three ATP are generated per revolution.
Bibliography
TEXTBOOK OF BIOCHEMISTRY Thomas M Devlin
Harpers Illustrated Biochemistry
Nomenclature Committee of the International Union of Biochemistry (NCIUB)
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