The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immo... more The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.
The thermohalophilic bacterium Rhodothermus marinus expresses a caa 3 -type dioxygen reductase as... more The thermohalophilic bacterium Rhodothermus marinus expresses a caa 3 -type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D-and K-proton channels, defined in most heme-copper ...
The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to ... more The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.
A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus.... more A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus. It is a complex with a minimum of three subunits (43, 27, and 18 kDa), containing five low-spin heme centers of the B and C types, in a 1:4 ratio. All the C-type hemes are in the same subunit (27 kDa). Three distinct redox transitions, at 235, 80, and -45 mV, were observed by visible redox titrations. The first involves one B- and one C-type hemes, and in the other two transitions one and two C-type hemes are involved, respectively. Spectroscopic data strongly suggest that the two hemes intervening in the last transition are in van der Waals contact, yielding a split Soret band. Electron paramagnetic resonance spectra of the oxidized complex show resonances of five low-spin ferric heme centers. Upon reduction with ascorbate, all these resonances vanish and a new one attributed to the last pair of hemes appears. A [3Fe-4S]1+/0 center copurifies with this complex, having a high reduction potential of +140 mV. No Rieske-type centers are detected in R. marinus and no effect is observed in the respiratory rates when the typical bc1 complex inhibitors are present, suggesting that such a complex is absent in R. marinus [Pereira et al. (1994) FEBS Lett. 352, 327-330]. The newly isolated cytochrome bc complex has quinol:cytochrome c or high-potential iron-sulfur protein (HiPIP) oxidoreductase activity, being a functional analogue of the canonical bc1 complexes; i.e., it is the complex III in R. marinus. This complex plays a central role in this bacterium's electron-transfer chain, coupling the electron transfer between the quinols reduced by the dehydrogenases and the HiPIP, the final electron donor to the terminal oxidases [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1276-1283].
The Rhodothermus marinus caa(3 )haem-copper oxygen reductase contains all the residues of the so-... more The Rhodothermus marinus caa(3 )haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278(I) in Paracoccus denitrificans aa(3)), being nevertheless a true oxygen reductase reducing O(2) to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256(I), R. marinus caa(3) numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278(I), as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa(3), using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa(3) oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa(3 )Tyr256(I) is not proton active at physiological pH, in contrast to the aa(3) Glu278(I) which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa(3) we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262(I) and Lys336(I), caa(3) numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.
A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus.... more A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus. It is a complex with a minimum of three subunits (43, 27, and 18 kDa), containing five low-spin heme centers of the B and C types, in a 1:4 ratio. All the C-type hemes are in the same subunit (27 kDa). Three distinct redox transitions, at 235, 80, and -45 mV, were observed by visible redox titrations. The first involves one B- and one C-type hemes, and in the other two transitions one and two C-type hemes are involved, respectively. Spectroscopic data strongly suggest that the two hemes intervening in the last transition are in van der Waals contact, yielding a split Soret band. Electron paramagnetic resonance spectra of the oxidized complex show resonances of five low-spin ferric heme centers. Upon reduction with ascorbate, all these resonances vanish and a new one attributed to the last pair of hemes appears. A [3Fe-4S]1+/0 center copurifies with this complex, having a high reduction potential of +140 mV. No Rieske-type centers are detected in R. marinus and no effect is observed in the respiratory rates when the typical bc1 complex inhibitors are present, suggesting that such a complex is absent in R. marinus [Pereira et al. (1994) FEBS Lett. 352, 327-330]. The newly isolated cytochrome bc complex has quinol:cytochrome c or high-potential iron-sulfur protein (HiPIP) oxidoreductase activity, being a functional analogue of the canonical bc1 complexes; i.e., it is the complex III in R. marinus. This complex plays a central role in this bacterium's electron-transfer chain, coupling the electron transfer between the quinols reduced by the dehydrogenases and the HiPIP, the final electron donor to the terminal oxidases [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1276-1283].
The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immo... more The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.
The thermohalophilic bacterium Rhodothermus marinus expresses a caa 3 -type dioxygen reductase as... more The thermohalophilic bacterium Rhodothermus marinus expresses a caa 3 -type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D-and K-proton channels, defined in most heme-copper ...
The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to ... more The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.
A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus.... more A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus. It is a complex with a minimum of three subunits (43, 27, and 18 kDa), containing five low-spin heme centers of the B and C types, in a 1:4 ratio. All the C-type hemes are in the same subunit (27 kDa). Three distinct redox transitions, at 235, 80, and -45 mV, were observed by visible redox titrations. The first involves one B- and one C-type hemes, and in the other two transitions one and two C-type hemes are involved, respectively. Spectroscopic data strongly suggest that the two hemes intervening in the last transition are in van der Waals contact, yielding a split Soret band. Electron paramagnetic resonance spectra of the oxidized complex show resonances of five low-spin ferric heme centers. Upon reduction with ascorbate, all these resonances vanish and a new one attributed to the last pair of hemes appears. A [3Fe-4S]1+/0 center copurifies with this complex, having a high reduction potential of +140 mV. No Rieske-type centers are detected in R. marinus and no effect is observed in the respiratory rates when the typical bc1 complex inhibitors are present, suggesting that such a complex is absent in R. marinus [Pereira et al. (1994) FEBS Lett. 352, 327-330]. The newly isolated cytochrome bc complex has quinol:cytochrome c or high-potential iron-sulfur protein (HiPIP) oxidoreductase activity, being a functional analogue of the canonical bc1 complexes; i.e., it is the complex III in R. marinus. This complex plays a central role in this bacterium's electron-transfer chain, coupling the electron transfer between the quinols reduced by the dehydrogenases and the HiPIP, the final electron donor to the terminal oxidases [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1276-1283].
The Rhodothermus marinus caa(3 )haem-copper oxygen reductase contains all the residues of the so-... more The Rhodothermus marinus caa(3 )haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278(I) in Paracoccus denitrificans aa(3)), being nevertheless a true oxygen reductase reducing O(2) to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256(I), R. marinus caa(3) numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278(I), as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa(3), using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa(3) oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa(3 )Tyr256(I) is not proton active at physiological pH, in contrast to the aa(3) Glu278(I) which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa(3) we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262(I) and Lys336(I), caa(3) numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.
A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus.... more A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus. It is a complex with a minimum of three subunits (43, 27, and 18 kDa), containing five low-spin heme centers of the B and C types, in a 1:4 ratio. All the C-type hemes are in the same subunit (27 kDa). Three distinct redox transitions, at 235, 80, and -45 mV, were observed by visible redox titrations. The first involves one B- and one C-type hemes, and in the other two transitions one and two C-type hemes are involved, respectively. Spectroscopic data strongly suggest that the two hemes intervening in the last transition are in van der Waals contact, yielding a split Soret band. Electron paramagnetic resonance spectra of the oxidized complex show resonances of five low-spin ferric heme centers. Upon reduction with ascorbate, all these resonances vanish and a new one attributed to the last pair of hemes appears. A [3Fe-4S]1+/0 center copurifies with this complex, having a high reduction potential of +140 mV. No Rieske-type centers are detected in R. marinus and no effect is observed in the respiratory rates when the typical bc1 complex inhibitors are present, suggesting that such a complex is absent in R. marinus [Pereira et al. (1994) FEBS Lett. 352, 327-330]. The newly isolated cytochrome bc complex has quinol:cytochrome c or high-potential iron-sulfur protein (HiPIP) oxidoreductase activity, being a functional analogue of the canonical bc1 complexes; i.e., it is the complex III in R. marinus. This complex plays a central role in this bacterium's electron-transfer chain, coupling the electron transfer between the quinols reduced by the dehydrogenases and the HiPIP, the final electron donor to the terminal oxidases [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1276-1283].
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