Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 11 April

© R. Paselk 2006
 
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Oxidative Phosphorylation

ATP Synthase (Complex V; F0F1 ATP synthase, Figure 14.14) [overhead]: ATP synthase uses the proton gradient to make ATP from ADP and Pi. 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 Figure 14.15: 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 Pi 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 Pi (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 Pi 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.

(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. Note that in this exchange ATP4- leaves the matrix as ADP3- 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 Pi 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 + Pi to ATP and one to transport ATP out of, while bringing ADP and Pi 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:

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.

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.

Fat Metabolism

Fats come from two main sources: stored body fat and dietary fat. Dietary fat must first be emulsified to increase its surface area for contact with the water soluble lipases. This occurs largely in the duodenum after mixing with the bile acids, a family of cholesterol derived detergents. Triacylglycerols can then be hydrolyzed by pancreatic lipase to free fatty acids and 2-monoacylglycerol:

The fatty acids and monoacylglycerol are absorbed by the intestinal cells, converted to fatty acyl CoA and reassembled into triacylglycerols. The triacyl glycerols then assemble with phospholipids and lipoproteins to form chylomicrons for transport through the lymph and blood to the tissues.

When the chylomicrons reach tissue cells the triacylglycerols are again hydrolyzed by lipoprotein lipase to fatty acids which can be taken up by the peripheral tissue cells. In adipose cells the fatty acids are then converted into fatty acyl CoA's and combined into triacylglycerols for storage. Alternatively the fatty acids can be broken down for energy using the beta-oxidation pathway.


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Last modified 11 April 2007