EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying.

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic approximately 9 nm F1 (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the "etched" surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with freeze-dried TU particles (trypsin-urea treated SMP) that are devoid of F1 (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-"etching."     

The inactivation of mitochondrial F1 ATPase by H2O2 is mediated by iron ions not tightly bound in the protein.

Exposure to purified mitochondrial F1 ATPase to continuous flux of H2O2 resulted in significant loss (up to 60%) of the ATP hydrolytic activity. The presence of chelating agents including desferrioxamine or previous selective removal of the iron ions not tightly bound in the protein completely prevented the inactivation, whereas re-loading of the enzyme with F3+ restored the sensitivity to H2O2. A marked protective effect was provided as well by mannitol or by Cu,Zn superoxide dismutase. The results indicated the decomposition of H2O2 by redox-active iron-protein adducts as responsible for the enzyme inactivation, probably through site-directed generation of more highly reactive oxygen species. A possible role for iron associated to F1 component in the oxidation, aging and turnover of ATP synthase complex in vivo may be suggested on the basis on these results.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.     

Synthesis of ATP by soluble mitochondrial F1 ATPase and F1-inhibitor-protein complex in the presence of organic solvents

The F1 and F1-inhibitor-protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20-50% v/v), ethylene glycol (20-60% v/v) or poly(ethylene glycol) 4000 and 8000 (30-50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents. In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1-inhibitor-protein there was no synthesis of ATP in the absence of MgCl2. The rate of ATP synthesis became faster as the MgCl2 concentration in the medium was raised from 0.1-10 mM. The Km for Pi of F1 was in the range of 0.8-1.5 mM. The Km for Pi of the F1-inhibitor-protein was much higher than that of F1 and could not be measured. In the presence of 10 mM MgCl2 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1-inhibitor-protein were 5.2-10.4 h-1 and 3.5-5.9 h-1 respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h-1. The tightly bound ATP of F1 and F1-inhibitor-protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgCl2 concentration was suddenly decreased. Both in presence and absence of Mg2+, 40-60% of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non-radioactive ATP was added to the assay medium. This was not observed when F1-inhibitor-protein was used.     

Thermal stability of soluble mitochondrial H+-ATPase.

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the alpha-helix of H+-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20 degrees--40 degrees C. Above 40 degrees C, the pattern of thermal decomposition of the three-dimensional structure of H+-ATPase is independent of the nature of nucleotides present. Highly stable alpha-helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Effect of pH and substitution of H2O for D2O on oxygen liberation by chloroplasts

The rate of dark relaxation of the oxygen evolving system in chloroplasts is shown to depend on the value of the surface charge of some chloroplast membrane component having protein nature and isoelectric point at pH 6.0. The substitution of H2O for D2O leads to isoelectric point shift of this protein.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Specificity of acidic phospholipids (CL & PA) in the activation of mitochondrial F0F1 ATPase by Mg2+

The interaction of Mg2+ with native F0F1 ATPase was studied. The hydrolytic activity of F0F1 ATPase could be competitively activated by Mg2+, but the preincubation of F0F1 ATPase with cholate eliminated the Mg2+ effect. The result from the comparison of the effect of Mg2+ on F0F1 ATPase with that on soluble F1 ATPase, and the fact that the activation of Mg2+ on cholate-treated F0F1 ATPase could be reconstituted only by divalent acidic phospholipid cardiolipin, indicate that there exists a specificity between the acidic phospholipids of the mitochondrial inner membrane and Mg2+ enhancement of ATP-hydrolyzing activity of F0F1 ATPase.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.