Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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The Electron Transport System, cont.

With these electron transport components we can create a path for the transfer of electrons from substrate to oxygen, as above, but we want more than a wire: want to use this flow of electrons to run a couple of "motors" to pump protons across the inner mitohondrial membrane, forming an electrochemical gradient, as shown in Figure 14.6, p 424 [slide]. Note that on this diagram the heights of the boxes indicates the change in standard potential or energy. From this we can see that complexes I and IV obviously have enough energy change to support the phosphorylation of ADP to ATP, while complex III is marginal and complex II obviously does not have sufficient energy change.

But how is this energy captured? Mitochondria appear to be using the energy of moving the electrons through this potential gradient to pump protons across the inner mitochondrial membrane. The resulting proton motive gradient can then be used to make ATP as we shall see later. Lets look at the four complexes and what seems to be occurring in each.

Complex I

cartoon of complex I

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[Figure 14.9, p 427] Note that the electrons are received as a pair (a hydride ion) on NADH, but most carriers can only handle single electrons so FMN acts as a transducer, picking up a pair of electrons, but passing them on singly to the FeS centers in this complex. The electrons are then passed singly on to CoQ, which, like FMN can carry either pairs or single electrons. Four H+ are pumped across the inner mitochondrial membrane by complex I. (Note that formally the protons from NADH and H+ can be considered as going to FMNH2 and then QH2.)

Complex II

cartoon of complex II

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[Figure 14.11, p 428] An electron pair enters this complex via FAD going to FADH2. Of course FAD has the same active portion as FMN so it can again carry electrons single or in pairs. FADH2 then passes its electrons on singly to FeS centers, which then pass them on to CoQ. No protons are pumped by this complex. Thus we see the difference in the number of ATPs which are produced by NADH vs. FADH2.

Coenzyme Q (ubiquinone)

Seen previously

Complex III

cartoon of complex III

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[Figure 14.14, p 429] This complex picks up single electrons from the CoQ pool, taking the quinol form in two steps to the quinone form of the coenzyme. One electron is passed on through FeS centers to Cytochrome c, while a second is recycled via cytochrome b (b566 & b560) to reduce Q to Q- in the Q-cycle. A second QH2 repeats the cycle and provides the second electron for the first recycled Q, passing another electron to cytochrome C. The net result is the oxidation of one QH2 and the transport of four H+ (two from the QH2 pool {thus Complex I or II} and two from the mitosol) for each pair of electrons flowing through the complex as shown in Figure 14.14, p 429 and Figure 14.16, p 430 [slide].

Cytochrome c

cytochrome c cartoon

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[Figure 14.15, p 430] This cytochrome is a mobile protein carrier attached to the outer side of the inner mitochondrial membrane (a peripheral membrane protein). It transports single electrons from complex III to complex IV.

Complex IV

cartoon of complex IV

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[Figure 14.19, p 432] This complex picks up electrons singly from cytochrome c and transfers them via cytochrome a-CuA to cytochrome a3-CuB where they are passed on to oxygen. Note that four electrons are needed for each oxygen molecule, and that the equivalent of two H+ are transferred out of the matrix for each pair of electrons (4 protons/oxygen). The high DeltaG for this complex, which would allow a significantly larger number of protons to be pumped, assures the completion of the overall reaction of oxygen and NADH to give water.

(For an insider's review and evidence on ETS and OxPhos see M. Saraste Science 283 (5 March 1999) pp 1488-93)

 

Oxidative Phosphorylation

Let's review how the energy captured of the ETS is initially stored and then used to make ATP (Chemiosmotic theory). Mitochondria appear to be using the energy of moving the electrons through this potential gradient to pump protons across the inner mitochondrial membrane. [Figure 14.1, p 418]

electron transport system cartoon

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The resulting proton motive gradient can then be used to make ATP. Note the consequences of the nature of this system in terms of ATP production vs. proton gradient and electron transport.

Let us predict the relative reduction potentials of key components of ETS assuming a lo [ADP]. Key components that are readily monitored (via spectrophotometry) are [components in the same complex are enclosed in brackets]:

[NADH right arrow FMN]Cmplx I right arrow Q right arrow [Cyt b right arrow Cyt c1]Cmplx III right arrow Cyt c right arrow [Cyt a right arrow Cyt a3]Cmplx IV right arrow O2

Low [ADP] will slow each of the Complexes where ATP is produced due to the backpressure of the proton gradient, that is, it will be harder to pump protons against a strong gradient, so it will be hard to transport electrons through the complexes. Thus we expect:

A simulated plot is shown below.

bar graph of simulated reduction levels of ets components

ATP Synthase

So how is ATP made from ADP and Pi? [Complex V; F0F1 ATP synthase, Figure14.22, p 433]: ATP synthase uses the proton gradient to make ATP from ADP and Pi.

ATP Synthase molecular model

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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 14.23, p 434: open, loose, or tight. [slide animation] 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.

ATPase mechanism

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(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.)

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Last modified 10 April 2013