The oxidation of NADH occurs in a stepwise manner involving an electron transport chain. Reducing equivalents go via a series of carriers to oxygen. Electrons flow downhill, energetically. Energy released in this process is used to generate the Proton Motive Force which is used by the cell to make ATP.

COMPONENTS OF THE ELECTRON TRANSPORT CHAIN (ETC)

The components of the ETC are membrane bound. Their redox potentials vary between NAD (Eo = -320mV) and O2 (+820mV). Note that the redox potentials of different cytochromes and flavoproteins etc. may differ even though they have the same prosthetic groups. The surrounding protein modifies the Eo of the heme and flavin groups. The mitochondrial ETC is fixed in composition and is longer and generates more energy than that of E. coli. In contrast, many bacteria, including E. coli have multiple alternative ETCs produced in response to different environmental conditions.

a) NADH Dehydrogenase. Found in the cytoplasmic membrane with other components of the ETC. E. coli contains two alternative NADH dehydrogenases: NADH-DH I contains one FAD, several FeS groups and one bound ubiquinone(UQ) per enzyme. This isoenzyme is coupled and generates PMF. Encoded by the gene nuo (NADH UQ oxidoreductase). Reducing equivalents go from NADH Æ FAD Æ FeS Æ UQ. NADH-DH II has FAD & UQ but contains no FeS clusters, and is not coupled and cannot generate PMF. Encoded by ndh. It is used when there is surplus NADH to be oxidized but the cells have enough energy. The mitochondrial enzyme resembles NADH I except that it has FMN instead of FAD.

b) Ubiquinone. All ETCs contain quinones. Quinones are hydrogen carriers and carry 2H at a time. The structure of the quinone nucleus varies: ubiquinone (respiration) & plastoquinone (photosynthesis) each have a 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. InE. coli n = 8 and in mitochondria n = 10.

There is more ubiquinone than all other ETCcomponents 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|

c) 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 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+

In E. coli, FeS groups are found in NADH-dehydrogenase-I and in the cytochrome b region of the chain. In mitochondria there are at least 7 FeS centers. Four are associated with mitochondrial NADH-dehydrogenase two are associated with cytochrome b and one with cytochrome c1.

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.

Flavoproteins These contain the yellow flavin coenzymes FAD & FMN which have already been discussed. 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. Note that E. coli contains two types of LDH:

1) Soluble, NAD-linked, converts pyruvate to lactate in fermentation

2) Membrane-bound, FAD-linked, oxidizes lactate to pyruvate in air and feeds into ETC. There are actually two FAD-linked isoenzymes, one each for the D- & L- isomers of lactate.

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.

e) Cytochromes. Electron carriers with Fe-porphyrin (heme) groups. Alternate between Fe3+ and Fe2+. The Fe in heme has six coordination positions, four of which are filled by N-atoms of the heme. 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 of the final cytochromes in the ETC which react with molecular oxygen. In mitochondria this is cyt a/a3, in E. coli both cyt d and cyt o are terminal oxidases. Cytochrome oxidases can bind CO, CN- or HS- (from H2S) instead of O2 and the enzyme activity is then killed.

In mitochondria and many obligately aerobic bacteria there is a full set of cytochromes: Electrons go from UQ to cyt b to cyt c1 to cyt c to cyt a/a3. Cyt a/a3 is known as cytochrome c oxidase since it oxidizes cyt c at the expense of molecular oxygen. Cyt a/a3 contains copper ions as well as heme.

In facultative anaerobes there is a shorter chain and cyt c & cyt c1 are both missing. Electrons go from UQ to cyt b to cyt o or d.

In E. coli high aeration gives mostly the cytochrome o complex:

2 heme b (b555 & b562) and 2 copper ions.

Low aeration results in appearance of the cytochrome d complex:

2 heme b (b558 & b595) and 2 heme d, but no copper.

The Km values for O2 are 0.2mM for cyt o and 0.02mM for cyt d which is consistent with the idea that cyt d is needed when the concentration of O2 is low. Thes are sometimes called ubiquinol oxidases since they oxidize UQ at the expense of molecular oxygen. There is no cytochrome c, c1 or a in the E.coli aerobic respiratory chain. Cyt d used to be called cyt a1 but is not really an a-type. A c-type cytochrome is found in the anaerobic nitrite reductase of E.coli.

Protoheme IX = Heme b Heme d [a chlorin]

Porphyrin = Heme without the Fe atom and with 2 H atoms instead. The four 5-membered rings each containing N are pyrrole rings. Cytochromes are classified according to the substituents at positions 2 and 4 of the porphyrin/heme ring:

Cytochrome b: vinyl groups at both 2 and 4 positions. This gives protoheme IX and is also found in hemoglobin, myoglobin, catalase and peroxidase. Cytochrome o of E. coli is a b-type.

Cytochrome c: positions 2 and 4 both are -CH(CH3)-S-Protein.

Cytochrome a: position 2 has a 17-carbon side chain, position 4 is vinyl and position 8 has -CHO (instead of the usual CH3).

Chlorins are hemes in which one or more of the pyrrole rings are reduced. Heme d of E.coli cytochrome d (= cyt a1) and siroheme of sulfite reductase are chlorin derivatives.

Originally, it was thought that ATP was made by direct chemical phosphorylation at 3 places along the ETC where sufficient energy was available from the drop in redox potential. These are the three coupling sites. It is now known that 2 protons are expelled at each coupling site, so generating the Proton Motive Force (PMF). ATP is made indirectly using the PMF as a source of energy. Each pair of protons yields one ATP. In E. coli and many bacteria there is no cytochrome c or c1 and thus coupling site #2 is missing so that only 2 ATP per NADH may be made.

Chemical coupling theory. Model is reactions such as glyceraldehyde phosphate dehydrogenase and pyruvate dehydrogenase where a redox reaction directly generates a high energy chemical intermediate, ie: diphosphoglyceric acid and acetyl-lipoate respectively. Disproven and of historical interest only.

Chemiosmotic theory. Proposed by Mitchell 1961. Finally proved by about 1977. Nobel Prize 1979. ATP synthesis is coupled indirectly to electron transport via the Proton Motive Force (PMF). In the chemiosmotic model, each of the coupling sites is responsible for extruding protons, so creating a proton concentration gradient across the membrane. The ATP synthase uses this gradient to energize the synthesis of ATP.

The membrane must be impermeable to H+ (and OH-). Since H+ is pumped out, the exterior will become acidified and the cell interior should become alkaline relative to the outside. In practice the internal pH is held constant at 7.5 approx. The proton gradient may be all or partially converted to a charge difference by exchanging potassium ions for protons. The PMF thus consists of two components: a pH difference (Delta pH) and a charge difference (or membrane potential, Delta Psi) across the membrane.

Stoichiometry. Original theory of Mitchell has 2H+ extruded per site with 3 sites per mitochondrion or 2 sites per E. coli and hence P:O ratios of 3:1 or 2:1 assuming 2H+ can drive formation of one ATP. (P:O ratio = ATP per oxygen atom consumed = ATP per 2 electrons sent down ETC). Some data suggests 3 or 4 protons may be extruded at certain sites, but this only applies to mitochondria, not to E. coli , and is not discussed here.

Mitchell's Z-Scheme. In Mitchell's original Z-scheme the ETC is arranged in a series of loops with hydrogen carriers (A & C) facing the inside of the bacterial cell or mitochondrion and electron carriers (B & D) facing outwards. Each hydrogen carrier receives two hydrogen atoms - 2[H] and it transfers 2e- to the following electron carrier. Since 2[H] = 2H+ + 2e- this leaves two protons (2H+) to dispose of and these are expelled so generating the PMF. The electron carrier transfers the electrons to the next hydrogen carrier. In order to get 2[H], two 2H+ must also be picked up from the inside of the bacterial cell. The ETC contains one proton extrusion loop for each coupling site.

Mitchells Equation

Proton chemical potential: m = 2.3RT(pHi - pHo) = 2.3RT Delta pH

Electrical potential: m = F(Psi i- Psi o) = F DeltaPsi

i refers to inside, o to outside, F = Faradays constant, Psi = charge

Electrochemical potential = m(chemical) + m(electrical):

m (H+) = F DeltaPsi - 2.3RT Delta pH.

Proton Motive Force:

PMF = m(H+)/F = DeltaPsi - 60 Delta pH

PMF is the proton motive force in millivolts. Conversion factor is approx. 60mV/pH unit at 37°C. Note that the relative contributions of DeltaPsi and Delta pH to PMF vary with the pH of the growth medium. Note also that PMF can only be maintained as long as membrane forms a sealed vesicle.

To generate 100mV of PMF requires the extrusion of about 60,000 protons/cell. The PMF generated by E. coli is 150-200 mV. How the intracellular pH is maintained at pH7.5 is unknown. It is known that at an external pH of 7.5 the Delta pH is replaced by a DeltaPsi by swapping K+ for H+. In effect, E. coli is a tiny living battery.

To convert ADP to ATP requires 7.3 kcal/mole energy input. This is equivalent to the energy stored in a pair of protons at a pH gradient of 3.5 pH units or at a charge difference of 240mV.

Uncouplers are proton ionophores(= ion carriers). They carry H+ across membranes and consequently allow the PMF to collapse. Both the protonated and unprotonated forms can diffuse. Protons are picked up on the outside and released on the inside. eg: 2,4-Dinitrophenol (2,4-DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the similar chloro compound CCCP.

Other Ionophores: Carriers exist for ions other than the proton eg:

Valinomycin carries potassium(K+) or rubidium(Rb+) ions and will therefore collapse the DeltaPsi but not the Delta pH

Nigericin exchanges Na+, K+ or Rb+ for H+ so collapsing Delta pH but not DeltaPsi ie opposite effect to valinomycin

Gramicidin is not a carrier but forms a channel in the membrane allowing through H+, Na+, K+, & Rb+ thus collapsing both Delta pH & DeltaPsi

Measurements of DeltaPsi and Delta pH.

Delta pH may be measured by the distribution of weak acids across the membrane. This depends on the Delta pH. Acids used eg: acetic acid, salicylic acid, DMO (5,5-dimethyloxazolidine-2,4-dione):

1) Membrane permeable to undissociated acid(HA) but not to H+ or A-

2) On each side of membrane HA ¥ H+ + A-

3) The extent of dissociation depends on pH

4) Thus extent of dissociation and hence the amount of HA differs on either side of membrane and this can be measured.

DeltaPsi may be measured indirectly by a similar method. A membrane permeable cation will be distributed according to DeltaPsi. For example, the ionophore valinomycin plus K+ or Rb+, or a lipophilic cation such as TPMP+ (triphenylmethylphosphonium) can be used.

DeltaPsi may be measured directly with a voltmeter and electrodes:

1) grow E. coli with mecillinam, a penicillin derivative which, in sublethal amounts, produces giant spherical cells (upto 6 microns diam).

2) use small electrodes! (0.08 to 0.2 microns at tip).

3) coat electrodes with phospholipid to get a good seal with the cell membrane - required to avoid PMF collapsing.

4) read DeltaPsi from voltmeter.

Example Proton Motive Force Values
Organism	pHo	-60DeltapH			Delta Psi		PMF
Fermentative
Staphylococcus lactis	5.0	58 (benzoate)			95 (dye)		158
Staphylococcus faecalis	5.0	54 (DMO)			170 (DDA)		224
Fermentative/Respiratory
Bacillus subtilis	5.0	120 (salicylate)			30 (TPMP)		150
Bacillus subtilis	7.5	0 (salicylate)			140 (TPMP)		140
Escherichia coli	5.5	72 (DMO)			57 (Rb+/Val)		129
Escherichia coli	7.0	38 (DMO)			122 (Rb+/Val)		160
Escherichia coli	5.5	120 (DMO)			100 (electrodes)		220
Escherichia coli	7.5	0 (DMO)			142 (electrodes)		142
Photosynthetic
Rhodospirillum rubrum
chromatophores* as these are inside out, polarity of PMF is reversed		72 (CH3NH4+)			50 (SCN-)		122
whole cell vesicles		36 (acetate)			70 (TPMP)		106
Halobacterium halobium	6.0	73 (DMO)			113 (TPMP)		186
Halobacterium halobium	8.0	8 (DMO)			151 (TPMP)		159

OXIDATIVE PHOSPHORYLATION

ATP synthesis is driven by the PMF. It can be demonstrated that only the PMF and the ATP synthase enzyme complex are required in the presence of sealed membrane vesicles. Note: Experiment is usually done "inside out" with the ATPase stuck to the outside.

1) Make liposomes (i.e., lipid vesicles)

2) Add purified ATPase and ADP and Pi

3) Add excess K+ plus Valinomycin to outside

4) Positive charge builds up on inside - an artificial PMF

5) The artificial DeltaPsi repels protons, derived from: H2O = H+ + OH-

4) Protons exit via ATP synthase, and ATP is made

ATPase and ATP synthase refer to the same enzyme - which direction the reaction goes depends on the conditions. The ATP synthase consists of the headpiece, or F1-complex, which sticks into the interior of the cell and the Fo-complex which is membrane-bound.

The catalytic a and b subunits of the F1-complex are responsible for synthesizing ATP. The Fo complex consists of 3 proteins a, b and c (sometimes referred to as c, y, w). Fo forms a proton channel through the membrane. Protein c is very hydrophobic (a "proteolipid") and probably acts as the channel while a and b control assembly and binding to the F1-headpiece. Fo alone makes membranes permeable to protons. Addition of F1 stops proton transport except to drive ATP synthesis. DCCD (dicyclohexyl carbodiimide) binds covalently to protein c and blocks the proton channel. Oligomycin inhibits the ATPase reaction of the catalytic F1 complex. Oligomycin halts the whole electron transport chain & ATP synthetic pathway when added alone. However, if both oligomycin and an uncoupler are added then the ETC runs but no ATP synthesis occurs.

Protein Ratio Gene Subunit MW Function

a 3 uncA F1 55kd catalytic

a (or c) 1 uncB Fo 30kd binds to F1

e 1 uncC F1 15kd regulation

b 3 uncD F1 50kd catalytic

c (or w) 10 uncE Fo 8kd proton channel

b (or y) 2 uncF Fo 17kd binds to F1

g 1 uncG F1 31kd structure of F1

d 1 uncH F1 20kd binds F1 to membrane

The precise mechanism of ATP synthesis is obscure. The simplest scheme for ATP synthesis postulates removal of an oxygen from inorganic phosphate by two protons from the outside thus producing water on the outside and ATP on the inside. The inorganic phosphate is closest to the proton channel with the ADP to the inside (see diagram for Mitchell's scheme). An alternative is the use of energy from the protons to alter the conformation of the catalytic a and b subunits. The high energy state then releases its energy by converting ADP to ATP.

ATP VERSUS THE FORCE

The ATPase can interconvert PMF and ATP. In the absence of a PMF the ATPase will hydrolyse ATP and generate PMF. Hence ATP and PMF are interconvertible. If an uncoupler is added to E. coli or a mitochondrion the PMF is dissipated. The ATPase then tries to re-create the PMF by hydrolyzing all the ATP. Oligomycin inhibits the ATPase and will prevent hydrolysis of ATP under these conditions just as it prevents synthesis of ATP from PMF.

ATP is used for biosynthesis and also drives certain transport systems. Arsenate inhibits ATP driven transport systems, but not those energized directly by the proton motive force. In eukaryotes the PMF is generated in the mitochomdria or chloroplasts and is all converted to ATP. In bacteria the PMF is used directly to energize several membrane bound operations:- 

PMF driven transport systems (discussed previously):

Energy linked transhydrogenase. Normally NADH is converted to ATP whereas NADPH is used as reducing power in biosynthesis. NADPH may be generated directly by certain metabolic pathways which generate NADPH instead of NADH. A proportion of the glucose (around 20%) goes via the pentose phosphate pathway instead of the Embden-Meyerhof pathway. In many bacteria the isocitrate dehydrogenase in the Krebs cycle also makes NADPH. Another source is the NADP linked malic enzyme.

However if their is a shortage of NADPH then it is possible to convert NADH to NADPH. This requires energy in order to shift the equilibrium in favor of NADPH. Energy linked transhydrogenase is energized directly by the PMF and converts NADH + NADP to NADPH + NAD. Mechanism unknown. It is particularly important in some photosynthetic organisms which do not have any glucose to send down the pentose pathway. It is present in E. coli but not important.

Flagellar Motion. E. coli is powered by a proton drive - just like the ships of the galactic empire (read the article by R2D2 in Scientific Galactican, August 3079). The M-ring of the basal body is located in the cytoplasmic membrane and rotates relative to the S- or stator ring of the basal body of the flagellum which is embedded in the cell wall. Proton acceptor groups (e.g., -NH2) are present on the rotor-M-ring and anionic groups (e.g., COO-) are located over the exit channels for protons (see diagram). Each time one H+ enters the input channel the ring turns a fraction of the revolution as the newly formed -NH3+ group is attracted towards the -COO- group.

ANAEROBIC RESPIRATION

Oxygen is the ultimate electron acceptor for the ETC when bacteria grow aerobically. However, bacteria can respire in the absence of oxygen if some other suitable oxidant is available to act as electron acceptor. Use of such alternative electron acceptors is known as anaerobic respiration. It requires a modified ETC in order to transport electrons to the new acceptor. E. coli can use nitrate (NO3-), amine oxides (R3NO), sulfoxides (R2SO), fumarate and nitrite (NO2-). Many sulfur compounds can be used by a variety of bacteria. "Sulfide fermentation" is not actually fermentation but is an example of anaerobic respiration with sulfate (SO42-) as the electron acceptor. "Methane fermentation" is also anaerobic respiration, in this case with carbon dioxide (CO2) as the electron acceptor.

Do not confuse anaerobic respiration with the oxidative metabolism of lithotrophs such as Nitrobacter. Oxygen has a more positive Eo than nitrite, therefore oxidation of nitrite by oxygen produces energy. Nitrate has a more positive Eo than 2[H] from NADH or formate. Therefore oxidation of NADH or formate by nitrate produces energy. Lithotrophs oxidize inorganic materials using O2. A substance with an Eo between O2 and NADH may be used either to oxidize NADH or to reduce O2. e.g. the NO3-/NO2- couple which has Eo of +420mV compared to +820mV for O2/H2O and -320mV for NAD+/NADH.

Anaerobic nitrate respiration: NO3- + 2[H] = NO2- + H2O

Nitrite oxidation by Nitrobacter NO2- + 1/2O2 = NO3-

Nitrate reductase ( NAR) of E. coli is a very large membrane bound protein (Total MW approx. 800,000). It is induced by nitrate and repressed by oxygen. When maximally induced it comprises upto 25% of the protein of the cytoplasmic membrane. Pyruvate formate lyase is present in anaerobic cells growing on nitrate and produces a lot of formate plus acetyl-CoA. Most of the acetyl-CoA is converted to acetate which is excreted. This is because the Krebs cycle is not complete during anaerobic growth because oxoglutarate dehydrogenase is not made. The formate is oxidized via formate dehydrogenase to give 2[H] plus CO2. The reducing equivalents travel via the cytochrome b of the FDH complex to a quinone. NADH produced in glycolysis can also be reoxidized by nitrate. The quinone used anaerobically is mostly menaquinone (Vitamin K2). However, when grown on nitrate, which is a good oxidant, a mixture of ubiquinone and menaquinone is found.

Nitrate reductase contains 4 subunits:

NarG protein x4 (= a) MW = 150,000, carries Mo & FeS groups

NarH protein x4 (= b) MW = 60,000, carries FeS groups

NarI protein (= g) cytochrome b, often lost on purification

NarJ protein membrane binding subunit

The Mo is present as part of the low molecular weight (approx 1000) molybdopterin cofactor. All Mo proteins share the same cofactor, (except for nitrogenase which has a different Mo/Fe cofactor).

Formate dehydrogenase is another large membrane protein. It contains molybdopterin, FeS groups and selenium. There are two types of FDH. The anaerobic respiratory FDH is found in anaerobic cells using nitrate or other alternative electron acceptors. The fermentative FDH is linked to hydrogenase and is produced anaerobically in the absence of alternative electron acceptors. The Se is present as selenocysteine ie cysteine in which sulfur has been replaced by selenium.

Most anaerobic reductases such as those for TMAO (trimethylamine oxide), DMSO (dimethyl sulfoxide) etc are molybdoproteins. Tungsten (W) is an analog of Mo. In the presence of tungstate (WO¢¤-) the processing of molybdate (MoO¢¤-) into molybdopterin is inhibited and nitrate reductase and other Mo enzymes are inactivated. Excess molybdate will remedy this i.e., tungstate is a competitive inhibitor. The two anaerobic reductases which do not contain molybdenum are nitrite reductase - a heme protein and fumarate reductase - a flavoprotein.

Fumarate Reductase (FRD) FRD is repressed by both oxygen and nitrate but induced by fumarate. FRD contains FAD and FeS groups. There are 4 subunits. A large flavoprotein, a medium sized FeS protein and two small membrane anchor proteins. FRD is connected via a cytochrome b to menaquinone. FRD cannot use ubiquinone, it has to use menaquinone. No molydenum or selenium are found in FRD. Note that fumarate is an organic molecule and the 2[H] go to saturate a double bond i.e., no water is formed as with nitrate, TMAO or DMSO etc.

Unlike O2 and nitrate which both generate two pairs of protons per 2e- which travel down the electron transport chain and hence yield 2ATP, fumarate gives only one pair of protons and only one ATP. When a cell changes from oxygen to fumarate as electron acceptor the PMF drops from around -200mV to approx. -100mV. (For nitrate the PMF is intermediate between these two).