Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2007
Lecture Notes: 7 December	© R. Paselk 2007
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Oxidative Phosphorylation

Last time we looked at the electron transport system in mitochondria and how it produces a Chemiosmotic gradient to capture energy. (text Figure 19-17) Today we will look at how this electro-chemical energy gradient is converted into ATP energy

ATP Synthase (Complex V; F₀F₁ ATP synthase, text Figure 19-23f, a-e): ATP synthase uses the proton gradient to make ATP from ADP and P_i. It is bound to the inner membrane and has a characteristic knob and stalk structure as seen in electron micrographs. It can be broken into two multi protein components: The F₁ component (the "knob") hydrolyses ATP when it is isolated by itself and is referred to as F₁ ATPase. The F₀ component is a membrane spanning proton channel. When the two components are linked the passage of protons through the channel is coupled to ATP synthesis.

According to the binding-change mechanism there are three sites in the alpha₃beta₃ oligomer of the knob. At any given time the three sites are in three different conformations, as shown in text Figure 19-24: open, loose, or tight. [overhead] Each site passes sequentially through the three conformations, apparently while physically rotating 120° for each change. Following one site: 1) ADP and P_i bind to the site in the open conformation. 2) Passage of 3 protons through the channel causes the alpha-beta oligomer to rotate 120° and change to the loose or L conformation, , holding the ADP and P_i (all three active sites to go to the next conformation simultaneously). 3) Passage of another 3 protons through the channel causes the alpha-beta oligomer to rotate 120° and change to the tight conformation with consequent condensation of ADP and P_i to ATP. 4) Passage of another 3 protons through the channel causes the alpha-beta oligomer to rotate 120° and change to the open conformation, releasing ATP. Note the net result of 3 protons/ATP.

The rotation of the ATPase has been visualized using fluorescence microscopy via fluorescent tagging. (text Figure 19-25)

(For an insider's review and evidence on ETS and OxPhos see M. Saraste Science 283 (5 March 1999) pp 1488-93; for ATPase 'motor' see Paul D. Boyer (18 Nov 1999) "What matkes ATP synthase spin?" Nature 402: 247-8.)

MITOCHONDRIAL TRANSPORT/COMMUNICATION

Given the requirement for a very tight inner-mitochondrial membrane in order to maintain the proton electrochemical gradient, how do important charged molecules such as ATP and NADH get across the membrane?

ADP & ATP are obviously among the most important substances to transport into and out of the mitochondria. Adenine nucleotide translocase exchanges matrix ATP for cytosolic ADP in their magnesium-free forms. (text Figure 19-26) Note that in this exchange ATP^4- leaves the matrix as ADP^3- goes in, resulting in a net loss of (1-) for the matrix. This increases the charge gradient across the membrane, and thus must be driven by the mitochondrial proton gradient. Of course P_i must also be transported across the membrane with ADP to make ATP in the matrix. This is accomplished using another transporter which co-transports a dihydrogen phosphate and a single proton in an electroneutral process. Note the addition of these two processes is equivalent to moving one proton from the cytosol to the matrix, costing the gradient one proton (moving a negative charge out is equivalent to moving a positive charge out).

The net cost of providing an ATP to the cytosol is thus four protons: three to convert ADP + P_i to ATP and one to transport ATP out of, while bringing ADP and P_i into, the matrix. This accounts for the theoretical yield of ATP: (10 H⁺/NADH)/(4 H⁺/ATP) = 2.5 ATP/NADH. (Note that this means that bacteria may get more ATP (up to 38 ATP's instead of the 32 expected in mammals).

Reducing Equivalent Shuttles: In aerobic metabolism NADH from glycolysis must be regenerated to NAD⁺ in the mitochondria. Two shuttles are important in different tissues/organisms for this process:

• Glycerol Phosphate Shuttle: This shuttle is found in insect flight muscles, which sustain very high rates of aerobic glycolysis, and fast twitch muscle and brain in humans. (text Figure 19-28) The NAD⁺ is actually regenerated in the cytosol by the Glycerol-3-P DH reaction, which reduces dihydroxyacetone-P to glycerol-3-P:

The glycerol-3-P is then reoxidized to dihydroxyacetone-P by Flavoprotein dehydrogenase. This enzyme is situated on the inner mitochondrial membranes outer surface. It uses FAD to oxidize the glycerol-3-P to dihydroxyacetone-P, passing the electrons to CoQ (note the similarity to succinate DH, except for the location on the outer instead of the inner surface of the membrane). The organism can thus get 1.5 ATP equivalents for this NADH.

• Malate-aspartate shuttle: (packet) This is the common shuttle in mammalian systems including liver, kidney and heart. It is both more complex and more efficient than the glycerol phosphate shuttle, yielding 2.5 ATP/NADH. It can be thought of as occurring in two phases: 1) transport of electrons from the cytosol to the mitosol, and 2) regeneration of the cytosolic oxaloacetate. In phase 1 oxaloacetate in the cytosol oxidizes NADH to NAD⁺ using malate DH. Malate is then transported across the inner membrane in exchange for an a-ketoglutarate by Dicarboxylate translocase. Malate is then oxidized back to oxaloacetate in the matrix by Kreb's Cycle malate DH to give NADH in the matrix. In phase 2 the oxaloacetate is transaminated to aspartate, taking a-ketoglutarate to glutamate, which are transported, and the process is balanced as in the figure.

Regulation of Mitochondrial Oxidative Phosphorylation

The ETS appears to be regulated largely by the availability of ADP and NADH. For most catabolic situations [ADP] will be the controlling factor. Note that the effects of [ADP] will integrate the regulation of TCA and Glycolysis with that of ETS.

Pathway Diagrams

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Last modified 7 December 2007