RESUMO
Upon separation of a crude preparation of beta subunit ("beta fraction") from mitochondrial F(1)-ATPase containing one equivalent of Fe(III) in the nucleotide-independent site (1Fe(III)-loaded MF(1)), Fe(III) is almost completely recovered. CD spectra show that "beta fraction" maintains the structural changes induced by Fe(III) in the whole enzyme. In accordance, EPR reveals that the Fe(III) site geometry is conserved in "beta fraction." Moreover, the EPR spectra of 1Fe(III)-loaded MF(1) and its "beta fraction" undergo similar changes of the line-shape upon Pi binding at the catalytic site, indicating that the Pi and Fe(III) are proximal on beta. Highly purified beta in nucleotide-free form binds 1mol of Fe(III)/mol of protein. MF(1) "freezed" by inhibitors with two beta in closed conformation and one beta in open or half-closed conformation binds 1mol of Fe(III)/mol of enzyme. Therefore, the Fe(III) site location in the unique beta subunit not adopting the closed conformation is proposed.
Assuntos
Compostos Férricos/metabolismo , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/química , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores Enzimáticos/farmacologia , Subunidades Proteicas , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismoRESUMO
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).
Assuntos
Peróxido de Hidrogênio/farmacologia , Ferro , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/química , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/química , Compostos Ferrosos/química , Radicais Livres , Micro-Ondas , Oxirredução , ATPases Translocadoras de Prótons/efeitos dos fármacosRESUMO
FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.
Assuntos
Inibidores Enzimáticos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacologia , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Animais , Bovinos , Dicicloexilcarbodi-Imida/farmacologia , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Hidrólise , Cinética , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Ligação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
Iron ions in the two iron centers of beef heart mitochondrial F1ATPase, which we have been recently characterized (FEBS Letters 1996, 379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H2O2, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H2O2 thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F1ATPase in a molar ratio 1:1 rapidly reacts with H2O2 mediating a 30% enzyme inactivation. These results support the presence in mitochondrial and thermophilic F1ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F1ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F1ATPase undergoes a higher inactivation (75%) upon exposure to H2O2. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dismutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H2O2.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Ferro/química , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/química , Sítio Alostérico , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Sítios de Ligação , Bovinos , Sequestradores de Radicais Livres/farmacologia , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxirredução , Estresse Oxidativo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/efeitos dos fármacos , Substâncias Redutoras/farmacologiaRESUMO
The binding Fe(III) to F1ATPase purified from beef heart mitochondria has been characterized by chemical analyses and EPR spectroscopy. F1ATPase binds 2 mol of Fe(III)/mol of protein selectively in the presence of saturating concentrations of ATP. In the absence of nucleotides or in the presence of either saturating ADP or limiting ATP concentrations, the enzyme binds 1 equivalent of Fe(III). F1ATPase pretreated with 5'-p- fluorosulfonylbenzoyladenosine, that selectively modifies the non-catalytic sites, binds only 1 mol of Fe(III)/mol of protein in the presence of either saturating ATP or ADP, Fe(III)-loaded F1ATPase containing either 1 or 2 equivalents of Fe(III) show identical EPR signals at g=4.3. The signals are not perturbed by the binding of nucleotides to the enzyme while they are altered by phosphate addition. These results indicate that F1ATPase contains two distinct Fe(III)-binding sites, which differ from nucleotide-binding sites, and that one of these sites is opened up for Fe(III) uptake by conformational changes induced by binding of ATP to the loose non-catalytic site.
Assuntos
Compostos Férricos/metabolismo , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/análise , Hidrólise , ATPases Translocadoras de Prótons/análiseRESUMO
Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.