Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

Return Exam 2

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. 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 in).

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 in humans. [Figure 14.26, p 437]. 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: This is the common shuttle in mammalian systems. 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 [Figure 14.27, p 438]. 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 -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 -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.

P/O Ratios and Their Determination

A useful measure of metabolism is the number of ATP's captured for each oxygen atom reduced to water (ATP/H₂O ) or the phosphate captured per oxygen atom = P/O.

Let's consider the P/O ratio for the conversion of citric acid to malate in the mitochondria.

Reaction	Energy Product	factor	ATP Equivalents (@2.5 ATP/NADH)	Oxygens consumed
Isocitrate DH	NADH	1	2.5	1
2-oxoglutarate DH Complex	NADH	1	2.5	1
Succinyl-CoA Synthetase	GTP	1	1	0
Succinate DH	FADH₂	1	1.5	1
TOTAL=			7.5	3
P/O = ATP/O =			7.5/3 = 2.5

Lipids

Recall the lipid definition: The portion of an organism which will partition into a non-polar solvent.

Types of Lipids:

Fatty acids: long chain carboxylic acids. .Three of the most common are:
- Palmitic acid:

Stearic acid: (Figure 11.3a)

Oleic acid: C9-C10 (Delta⁹double bond)

Triacylglycerols: Three fatty acids esterified to glycerol. (Figure 11.6)
Glycerophospholipids: Two fatty acids and a phosphate esterified to glycerol. (Table 11.2):

glycerophospholipoid structure