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Mitchell's proton pumping loops

The reduction of a H-carrier by an electron carrier in one phase would lead to uptake of H⁺; the diffusion of the H-carrier (as a neutral species) across the membrane, and its oxidation on the other side by an electron carrier would lead to release of a H⁺; electron transfer back across the membrane would carry a -ve charge across the membrane in the opposite direction, thus allowing the loop to carry 1 H⁺ (net) in an electrogenic proton pumping loop. The picture below shows the Mitchellian proton pumping loops in the best defined electron transfer chain, that for the photosynthetic apparatus of Rb. sphaeroides.

The photosynthetic chain of Rhodobacter sphaeroides, shown as two Mitchellian proton pumping loops. The Rb. sphaeroides chain has all components represented by models of the active components based on X-ray crystallographic structures at atomic resolution, - the first supramolecular machine defined at the atomic scale.
Left: The photochemical reaction center, surrounded by the light harvesting complex, LH1; cytochrome c is docked against the reaction center.
Right: A dimer of the bc₁ complex containing two monomers, each with the three catalytic subunits.
The electrogenic (white arrows) and neutral arms (blue arrows) of the Mitchellian proton pumps are shown, with the uptake and release of protons (red arrows) at the catalytic sites. 

Physical chemistry of coupling between electron transfer and H⁺-transport

Phosophlipid membranes

Generalized proton circuit

The transport of ATP, ADP and phosphate across the mitochondrial membrane

1. The adenine nucleotide transporter.

This enzyme catalyses the exchange of ATP for ADP across the inner mitochondrial membrane. The charges on the substrates are such that ATP carries a net excess charge of -1 compared to ADP.

 



The exchange reaction is therefore electrogenic, and driven by the electrical component (Dy) of the proton gradient, so that entry of ADP and exit of ATP are favored.

 

In, or N-phase  Out, or P-phase

The thermodynamics of this process, are discussed in more detail here.

 

2. The phosphate transporter.

The phosphate transporter was the first of the mitochondrial metabolite transport systems to be demonstrated (Chappell and Crofts, 1965). The enzyme catalyses the net transfer of H₃PO₄ across the membrane in a neutral process. Since the predominant ionic species present at neutral pH is H₂PO₄^-, the mechanism must involve either exchange of H₂PO₄^- for OH^-, or cotransport of H₂PO₄^- with H⁺.

 

H₂PO₄^-_L + OH^-_R <==> H₂PO₄^-_R + OH^-_L or H₂PO₄^-_L + H⁺_L <==> H₂PO₄^-_R + H⁺_R

The exchange allows the chemical component of the proton gradient (-ZDpH) to drive uptake of phosphate into the mitochondrion.

 More general aspects of the thermodynamics of transport are covered here.

Ionophores, and transport of ions and metabolites by mitochondria

1. Transport of bivalent cations.

When a proton gradient is generated by coupled electron transfer or ATP-hydrolysis, mitochondrial take up Ca²⁺ from the external medium. The Ca²⁺-uptake is catalysed by a Ca²⁺ carrier in the inner membrane which exchanges Ca²⁺ for H⁺ (net electrogenicitiy, 1 + charge). When phosphate is also present, a net uptake of calcium phosphate occurs, which precipitates inside the mitochondria as hydroxyapatite, visible as dense granules in electron micrographs. If a weakly acid anion like acetate is present in the suspending medium, calcium acetate is accumulated, and the mitochondria swell osmotically because of the increased soluble salt inside.

 Transport of Ca²⁺ plays a physiological role in those cell processes which are activated or inhibited by changes in Ca²⁺ activity in the cytoplasm. The mitochondrial transport has a K_m in the physiological range, so uptake buffers the internal [Ca²⁺].
Mitochondria will transport Mn²⁺ or Sr²⁺ in place of calcium.

2. Metabolite transport.


Mitochondria have diverse metabolic functions, which vary from tissue to tissue. In liver mitochondria, a wide variety of externally added substrates can be processed by the mitochondrial metabolic machinery. The enzymes are predominantly in the matrix, requiring that substrates be transported across the mitochondrial inner membrane. The transport is catalysed by a superfamily of proteins, which show strong sequence homology across the whole spectrum of the eukaryote world, but no obvious prokaryote precursor. The metabolite transporters generally catalyse neutral exchanges, but some are electrogenic, or involve H⁺ cotransport or OH^- exchange, so that the transport equilibrium is determined by the proton gradient.
Transport of metabolites will be covered in greater detail later.

 

3. Ion transport catalysed by ionophores.


Characterization of the mechanisms of ionophores played a crucial role in our understanding of the proton gradient, and its role in chemiosmotic coupling. Four ionophoric mechanisms have proved particularly useful:
1. Valinomycin.

Valinomycin catalyses the transprt of K⁺ (and other monovalent cations of similar radius like Rb⁺ or Cs⁺, but not Na⁺) as a charged species across biological membranes (or hydrophobic phases in general). Transport is by a carrier mechanism. As a consequence, the electrochemical potential for the transported species is equal across the membrane, and the equilibrium condition applies.

The distribution of ions (usually isotopically labelled Rb⁺ is use) between inner and outer phases in the presence of valinomycin has been a favorite method for measuring membrane potential.
The mechanism of valinomycin will be discussed further in the context of transport.

2. Nigericin.


Nigericin catalyses the neutral exchange of monovalent cations across biological membranes, with a broad specificity. If a single cation predominates, the cation is exchanged for H⁺. As a consequence, when nigericin is added to mitochondria in the presence of excess K⁺, the -ZDpH component of the proton gradient is collapsed. The mechanism of nigericin is discussed in greater detail here.
3. Gramicidin.

Gramicidin catalyses an electrogenic flux of small monovalent cations, including protons, across biological membranes through a pore formed by two molecules end-to-end spanning the membrane. The gramicidin pore is much studied, and will be discussed in greater detail later in the context of transport.
As a consequence of the mechanism, gramicidin acts as an uncoupling agent. In the presence of excess monovalent cation (such as K⁺), the transport of K⁺ is favored over the leak or H⁺, and gramicidin will catalyse K⁺-uptake by mitochondria, similar to that seen with valinomycin.
4. Protonophores.

Mitchell proposed that uncouplers act by short-circuiting the proton gradient,- acting as proton conductors. This protonophoric activity has been much investigated, and the demonstration of a strong correlation between protonophoric activity in artificial membrane systems, and uncoupling activity, was a strong piece of evidence favoring the chemiosmotic hypothesis.
Detailed discussion of these useful reagents will be found in Lecture 14.

Respiratory control

In the absence of reactions to use the proton gradient, the rate of electron transfer is slower by a factor of ~10 than in the presence of ADP + phosphate, or an uncoupler (the RC ratio is usually ~10, though the value depends on substrate). In a system without leaks, the electron transport chain, the proton gradient, and the ATP synthase reaction would come to equilibrium, and there would be no flux. The residual electron transfer is determined by the rate at which protons leak back across the membrane through non-specific pathways, or the rate at which the various proton pumps slip the "clutches" which couple their mechanisms to the gradient.

Reversibility of the coupled system,- reversed electron transport

Equations for transport, chemical and electrochemical potential gradients