Mitochondria states

The mitochondrial respiratory chain consists of three proton pumps which act in series with respect to the electron flow and in parallel with respect to the proton circuit (Fig. 2.2a). Two limiting states are frequently referred to for isolated mitochondria - State 4 in which the proton current is limited by the inhibition of proton re-entry through the ATP synthase (due to either actual inhibition of the synthase or to the attainment of equilibrium), and State 3 in which there is ready proton re-entry into the matrix and hence brisk respiration. The State 3 condition can be due to an induced proton leak in the membrane or to the maintenance of AG tp below that required to equilibrate with AfiH+ (by either removing ATP, or following the addition of ADP. 

It is remarkable that reported rates of respiration per mg of protein can vary between different laboratories for mitochondria from the same source by a factor as large as 3-4. This may be partially due to differences in protein determination. A more appropriate point of reference is, therefore, the content of respiratory chains. The respiratory rate with natural substrates is maximally approx. 30 e /s aa in rat liver mitochondria (State 3u with succinate M. Wikstrom, unpublished results). The rate in State 4 is lower by about one order of magnitude. The maximum respiratory velocity of a 900 nm membrane segment is then 60 e /s and the average turnover time of cytochrome c and ubiquinone, 50 and 500 ms, respectively (cf.. Ref. 84). In spite of the lower mobilities applied here, it seems that lateral diffusion of cytochrome c and ubiquinone is still sufficiently fast to be compatible even with maximal rates of respiration with natural substrates. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

In common with cholesterol synthesis described in the next section, fatty acids are derived from glucose-derived acetyl-CoA. In the fed state when glucose is plentiful and more than sufficient acetyl-CoA is available to supply the TCA cycle, carbon atoms are transported out of the mitochondrion as citrate (Figure 6.8). Once in the cytosol, citrate lyase forms acetyl-CoA and oxaloacetate (OAA) from the citrate. The OAA cannot re-enter the mitochondrion but is converted into malate by cytosolic malate dehydrogenase (cMDH) and then back into OAA by mitochondrial MDH (mMDH) Acetyl-CoA remains in the cytosol and is available for fatty acid synthesis. 

Allen s theory of redox poise, and the evidence supporting it, are discussed in Chap. 3 of this volume. Here, I want to make a few general observations on necessity and workability. Each mitochondrion needs a genome because the speed of electron flow down the respiratory chains depends not just on supply and demand (concentration of NADH, 02, ADP and inorganic phosphate) but also on the number and redox state of respiratory complexes (Allen 1993,... 

Degree of Reduction of Electron Carriers in the Respiratory Chain The degree of reduction of each carrier in the respiratory chain is determined by conditions in the mitochondrion. For example, when NADH and 02 are abundant, the steady-state degree of reduction of the carriers decreases as electrons pass from the substrate to 02. When electron transfer is blocked, the carriers before the block become more reduced and those beyond the block become more oxidized (see Fig. 19-6). For each of the conditions below, predict the state of oxidation of ubiquinone and cytochromes b, clt c, and a + a3. 

Step A, the conversion of pyruvate to phosphoenolpyruvate, is accomplished by a circuitous process commencing with pyruvate entering the mitochondrion, which for gluconeogenesis to occur must be in a high-energy state. Under these conditions, the mitochondrial enzyme pyruvate carboxylase catalyzes the conversion of pyruvate to oxaloacetate-. 

Moore then explained how mitochondria are biological fuel cells. The oxygen reduction taking place in a mitochondrion is exactly the same as in a standard fuel cell. Using several enzymes and only earth-abundant elements, the mitochondrion converts electrochemical potential to biochemical work with efficiency greater than 90 percent. This is a steady-state process in which protons are pumped across the membrane to maintain its electrical potential. If... 

The control of the respiration process and ATP synthesis shifts as the metabolic state of the mitochondria changes. In an isolated mitochondrion, control over the respiration process in state 4 is mainly due to the proton leak through the mitochondrial inner membrane. This type of control decreases from state 4 to state 3, while the control by the adenine nucleotide and the dicarboxylate carriers, cytochrome oxidase, increases. ATP utilizing reactions and transport activities also increase. Therefore, in state 3, most of the control is due to respiratory chain and substrate transport. 

Fig. 2 Metabolism of superoxide radical and nitric oxide in the mitochondrial matrix. The numbers below the symbols indicate approximate steady state concentrations for mammalian organs under physiological conditions. The arrows reaching outside the mitochondrion indicate diffusion of HjOj and NO to the cytosol. QBP ubiquinone binding protein, NADH-DH NADH dehydrogenase...

Intramitochondrial steady state concentrations of NO were calculated as 20-50 nM NO [ 12] and a release of 29 nM NO was electrochemically measured after supplementation of a single mitochondrion with Ca-+ [18]. Under physiological conditions the tissues are oxygenated in the range of 20 pM Oj, with [O2]/[NO] ratios of 500-1000, which should competitively inhibit cytochrome oxidase by 16-26% [19]. 

After synthesis In the cytosol, the soluble precursors of mitochondrial proteins (Including hydrophobic Integral membrane proteins) interact directly with the mitochondrial membrane. In general, only unfolded proteins can be imported Into the mitochondrion. Chaperone proteins such as cytosolic Hsc70 keep nascent and newly made proteins in an unfolded state, so that they can be taken up by mitochondria. Import of an unfolded mitochondrial precursor is initiated by the binding of a mitochondrial targeting sequence to an import receptor in the outer mitochondrial membrane. These receptors were first identified by experiments in which antibodies to specific proteins of the outer mitochondrial membrane were shown to inhibit protein import into... 

Two major messengers feed information on the rate of ATP utilization back to the TCA cycle (a) the phosphorylation state of ATP, as reflected in ATP and ADP levels, and (b) the reduction state of NAD, as reflected in the ratio of NADH/NAD. Within the cell, even within the mitochondrion, the total adenine nucleotide pool (AMP, ADP, plus ATP) and the total NAD pool (NAD plus NADH) are relatively constant. Thus, an increased rate of ATP utilization results in a small decrease of ATP concentration and an increase of ADP. Likewise, increased NADH oxidation to NAD by the electron transport chain increases the rate of pathways producing NADH. Under normal physiological conditions, the TCA cycle and other... 

Jakovcic et al. (1971) claimed that the asynchronous increase in activity of the several enzymes does not conflict with the notion that the mitochondrion turns over as a unit. Turnover has usually been studied under steady state conditions of synthesis and degradation, and such conditions may not be applicable diuring periods of rapid growth and differentiation either in fetal cells or in their organelles. Indeed, Neubert et al. (1968) have stated that the faster the growth rate of a tissue, the longer the half-life of mitochondria thus, in a very rapidly growing tissue there is almost no turnover. 

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