Electron Transport
5.2 Th e Electron Carriers
The electrons fl ow through a series of electron carriers. Some of these carry hydrogen as well as electrons, and some carry only electrons. The electron carriers are as follows:
1. Flavoproteins (hydrogen and electron carriers) 2. Quinones (hydrogen and electron carriers) 3. Iron–sulfur proteins (electron carriers) 4. Cytochromes (electron carriers)
The quinones are lipids, whereas the other electron carriers are proteins, which exist in multiprotein enzyme complexes called oxi- doreductases. (See note 3 for examples of oxi- doreductases.) The electrons are not carried in the protein per se, but in a nonprotein molecule bound to the protein. The nonprotein portion that carries the electron is called a prosthetic group. (See note 4 for useful defi nitions.) The prosthetic group in iron–sulfur proteins is a cluster of iron–sulfi de, which is abbreviated as FeS. The prosthetic group in fl avoproteins (Fp) is a fl avin, which can be either fl avin adenine dinucleotide (FAD) or fl avin mononucleotide (FMN). The prosthetic group in cytochromes is heme. The chemistry of the prosthetic groups is described in Section 5.2.1. Some of the pros- thetic groups (fl avins) carry hydrogen as well as electrons, and they are referred to as hydrogen carriers. The quinones are also hydrogen car- riers. Some of the prosthetic groups (FeS and heme) carry only electrons, and they are referred to as electron carriers.
Each of the electron carriers, described in Sections 5.2.1 through 5.2.4, has a different electrode potential, and the electrons are trans- ferred sequentially to a carrier of a higher poten- tial to the fi nal acceptor, which has the highest potential.
The standard potentials at pH 7 of the elec- tron carriers and other electron donors and
two ring nitrogens. There are many different fl avoproteins, and they catalyze diverse oxida- tion–reduction reactions in the cytoplasm, not merely those of the electron transport chain in the membranes. Although all the fl avoproteins have FMN or FAD as their prosthetic group, they catalyze different oxidations and have dif- ferent redox potentials. These differences are due to differences in the protein component of the enzyme, not in the fl avin itself.
5.2.2 Quinones
Quinones are lipid electron carriers. Owing to their hydrophobic lipid nature, some are believed to be highly mobile in the lipid phase of the membrane, carrying hydrogen and elec- trons to and from the complexes of protein electron carriers that are not mobile. Quinone structures and oxidation–reduction reactions are shown in Fig. 5.3. All quinones have hydro- phobic isoprenoid side chains that contribute to their lipid solubility. The number of isoprene units varies but is typically 6 to 10. Bacteria make two types of quinone that function dur- ing respiration: ubiquinone (UQ), a quinone also found in mitochondria, and menaquinone (MQ, or sometimes MK). Menaquinones (Fig. 5.3C), which are derivatives of vitamin K, differ from ubiquinones in being naphthoquinones in which the additional benzene ring replaces the two methoxy groups present in ubiquinones (Fig. 5.3A,B). Menaquinones also have a much lower electrode potential than ubiquinones and are used predominantly during anaerobic res- piration, where the electron acceptor has a low potential (e.g., during fumarate respiration). A third type of quinone, plastoquinone (Fig. 5.3D), occurs in chloroplasts and cyanobacteria, and functions in photosynthetic electron transport. In plastoquinones, the two methoxy groups are replaced by methyl groups.
5.2.3 Iron–sulfur proteins
Iron–sulfur proteins contain nonheme iron and usually acid-labile sulfur (Fig. 5.4). The term “acid-labile sulfur” means that when the pH is lowered to approximately 1, hydrogen sulfi de is released from the protein. This is because there is sulfi de attached to iron by bonds that are rup- tured in acid. Generally, the proteins contain clusters in which iron and acid-labile sulfur are present in a ratio of 1:1. However, there may acceptors in metabolic pathways are shown in
Table 5.1.
5.2.1 Flavoproteins
A fl avoprotein (Fp) is an electron carrier that has as its prosthetic group an organic molecule called a fl avin. The term is derived from the Latin word fl avius, which means yellow, in ref- erence to the color of fl avins. The fl avins FAD and FMN are synthesized by cells from the vita- min ribofl avin (vitamin B2).
What is the difference between FMN and FAD? This is shown in Fig. 5.2. Phosphorylation of ribofl avin at the ribityl 5′-OH yields FMN, and adenylylation (addition of ADP) yields FAD.
As Fig. 5.2 illustrates, when fl avins are reduced they carry 2H• (equivalent to two electrons and two protons), one on each of
Table 5.1 Standard electrode potentials at pH 7
Couple Potential (mV) Fdox/Fdred (spinach) –432 CO2/formate –432 H+/H 2 –410 Fdox/Fdred (Clostridium) –410 NAD+/NADH –320
FeS (ox/red) in mitochondria –305 Lipoic/dihydrolipophilic –290 Sº/H2S –270 FAD/FADH2 –220 Acetaldehyde/ethanol –197 FMN/FMNH2 –190 Pyruvate/lactate –185 Oxaloacetate/malate –170 Menaquinone (ox/red) –74 cyt b558 (ox/red) –75 to –43 Fumarate/succinate + 33 Ubiquinone (ox/red) +100 cyt b556 (ox/red) +46 to +129 cyt b562 (ox/red) +125 to +260 cyt d (ox/red) +260 to +280 cyt c (ox/red) +250
FeS (ox/red) in mitochondria +280
cyt a (ox/red) +290
cyt c555 (ox/red) +355
cyt a3 (ox/red) in mitochondria +385 NO− 3/NO − 2 +421 Fe3+/Fe2+ +771 O2 (1 atm)/H2O +815
Sources: Thauer, R. K., K. Jungermann, and K. Decker.
1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 41:100–180. Metzler, D. E. 1977.
Biochemistry: The Chemical Reactions of Living Cells.
be more than one iron–sulfur cluster per pro- tein. For example, in mitochondria the enzyme complex that oxidizes NADH has at least four FeS clusters (see later, Fig. 5.9). The FeS clusters have different Eh values, and the electron trav- els from one FeS cluster to the next toward the higher Eh. It appears that the electron may not be localized on any particular iron atom, and the entire FeS cluster should be thought of as carrying one electron, regardless of the number of Fe atoms.
Iron–sulfurs proteins also contain cysteine sulfur, which is not acid labile, and bonds the iron to the protein. There are several different types of iron–sulfur protein, and these catalyze
Fig. 5.2 Structures of ribofl avin (X = H), FMN (X = PO3H2), and FAD (X = ADP). For convenience, the reduc- tion reaction is drawn as proceeding via a hydride ion even though this is not the actual mechanism in all fl avin reductions.
Fig. 5.3 The structure of quinones: (A) oxidized ubiquinone, (B) reduced ubiquinone, (C) oxidized menaqui- none, and (D) oxidized plastoquinone. The value of n can be 4 to 10 and is 8 for both quinones in E. coli. In
E. coli ubiquinone plays a major role in aerobic and nitrate respiration, whereas menaquinone is dominant
during fumarate respiration. One reason for this is that ubiquinone has a potential of +100 mV, versus +30 mV for fumarate. It is therefore at too high a potential to deliver electrons to fumarate. Menaquinone has a low potential, –74 mV, and is thus able to deliver electrons to fumarate. Plastoquinone is used in chloroplast and cyanobacterial photosynthetic electron transport.
Fig. 5.4 Scheme for FeS clusters: this is a Fe2S2 clus- ter. More than one cluster may be present per pro- tein. The sulfur atoms held only by the iron are acid labile. The iron is bonded to the protein via sulfur in cysteine residues.
oxygen. (In naming cytochromes, sometimes the O2-binding heme is given the subscript 3.)7
As the names imply, each cytochrome contains two types of heme, one being heme b and the other being heme d or o.7 As mentioned previ-
ously, the hemes can be distinguished according to the side groups that they possess, as summa- rized in Fig. 5.5. For example, heme o differs from heme b in having an hydroxyethylfarnesyl group substituted for a vinyl group. However, the only difference between heme b and heme c is that the latter is covalently bound to pro- tein by thioether linkages between the two vinyl groups and cysteine residues in the protein.
Hemes can usually be distinguished spectro- photometrically. When cytochromes are in the reduced state, absorption by the heme produces characteristic light absorption bands in the vis- ible range: the α, β, and γ bands. The α bands absorb light between 500 and 600 nm, the β bands absorb at a lower wavelength, and the γ bands (also called Soret bands) are in the blue region of the spectrum. The spectrum for a cyto- chrome c is shown in Fig. 5.6. Cytochromes are distinguished, in part, by the position of the max- imum in the α band. For example, cyt b556 has a peak at 556 nm and cyt b558 a peak at 558 nm.
Reduced minus oxidized spectra
Because of light scattering and nonspecifi c absorption, it is very diffi cult to resolve the different peaks of individual cytochromes in whole cells unless one employs difference spec- troscopy. For difference spectroscopy, the cells are placed into two cuvettes in a split-beam spectrophotometer, and monochromatic light from a single monochromator scan is split to pass through both cuvettes. In one cuvette the cytochromes are oxidized by adding an oxi- dant, and in the second cuvette they are reduced by adding a reductant. The spectrophotometer subtracts the output of one cuvette from the other to give a reduced minus oxidized differ- ence spectrum. In this way nonspecifi c absorp- tion and light scattering are eliminated from the spectrum, and the cytochromes in the prepara- tion are identifi ed.
Dual-beam spectroscopy
A dual-beam spectrophotometer is used to fol- low the kinetics of oxidation or reduction of a particular cytochrome. The instrument has numerous oxidation–reduction reactions in the
cytoplasm as well as in the membranes. (See note 5 for more information on iron–sulfur proteins.) The iron–sulfur proteins have char- acteristic electron spin resonance (ESR) spectra because of an unpaired electron in either the oxidized or reduced form of the FeS cluster in different FeS proteins. (See note 6 for a descrip- tion of electron spin resonance.) The iron–sulfur proteins cover a very wide range of potentials, from approximately –400 mV to +350 mV. They therefore can carry out oxidation–reduc- tion reactions at both the low-potential end and the high-potential end of the electron transport chain, and indeed are found in several locations. In the FeS cluster shown in Fig. 5.4, note that each Fe is bound to two acid-labile sulfurs and two cysteine sulfurs. This would be called an Fe2S2 cluster.
5.2.4 Cytochromes
Cytochromes are electron carriers that have heme as the prosthetic group. Heme consists of four pyrrole rings attached to each other by methene bridges (Fig. 5.5). Because hemes have four pyrroles, they are called tetrapyrroles. Each of the pyrrole rings is substituted by a side chain. Substituted tetrapyrroles are called porphyrins. Therefore, hemes are also called porphyrins. (An unsubstituted tetrapyrrole is called a porphin.) Hemes are placed in differ- ent classes, described shortly, on the basis of the side chains attached to the pyrrole rings. In the center of each heme there is an iron atom that is bound to the nitrogen of the pyrrole rings. The iron is the electron carrier and is oxidized to ferric or reduced to ferrous ion during elec- tron transport. Cytochromes are therefore one- electron carriers. The Eh values of the different cytochromes vary depending on the protein and the molecular interactions with surrounding molecules.
Classes of cytochromes
Figure 5.5 shows fi ve classes of heme that dis- tinguish the cytochromes: hemes a, b, c, d, and o. Hemes d and o have been found only in the prokaryotic cytochrome oxidases. Bacterial cytochromes include cytochromes bd (some- times called cytochrome d) and bo (some- times called cytochrome bo3 or cytochrome o), which are quinol oxidases that reduce
two monochromators: light from one is set at a wavelength at which absorbance will change during oxidation or reduction, and the second beam of light is at a nearby wavelength for which absorbance will not change. The light is sent alternatively from both monochromators through the sample cuvette, and the difference in absorbance between the two wavelengths is automatically plotted as a function of time.
Table 5.1 shows standard electrode potentials at pH 7 (E′0) of some electron donors, acceptors, and electron carriers. Notice that redox couples are generally written in the form “oxidized/ reduced.” Many of the oxidation–reduction reactions in the electron transport chain can be reversed by the ∆p as discussed in Section 4.7.1. This means that the ox/red ratio for several of the electron carriers (fl avoproteins, cytochromes,
Fig. 5.5 The prosthetic groups of the different classes of cytochromes. The hemes vary according to their side groups. Heme c is covalently bound to the protein via a sulfur bridge to a cysteine residue on the protein.
Four complexes can be recognized in mitochondria. They are complex I (NADH– ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (ubiquinol–cytochrome c oxidoreductase, also called the bc1 complex), and complex IV (cytochrome c oxidase, which is cytochrome aa3). Complexes I, III, and IV are coupling sites (Section 5.5). Each complex can have several proteins. The most intricate is complex I, from mammalian mitochondria, which has about 40 polypeptide subunits, at least four iron–sul- fur centers, one fl avin mononucleotide (FMN), and one or two bound ubiquinones. Analogous complexes have been isolated from bacteria, but in some cases (e.g., NADH–ubiquinone oxidoreductase and the bc1 complex), they have fewer protein components.8–11 (See note
12 for a description of complex II and how it varies with different bacteria.) Note the pat- tern in the arrangement of the electron carri- ers in mitochondria; a dehydrogenase complex accepts electrons from a primary donor and transfers the electrons to a quinone. The qui- none then transfers the electrons to an oxi- dase complex via intervening cytochromes. As described next, the same general pattern exists in bacteria.