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P Adelroth

Publications and source records attributed to P Adelroth.

30 records · Page 2Linked to original sources
PubMed1998

Proton-controlled electron transfer in cytochrome c oxidase: functional role of the pathways through Glu 286 and Lys 362.

We have used a combination of site-directed mutagenesis and spectroscopic techniques to investigate electron-transfer reactions between hemes a and a3 in cytochrome c oxidase. A state of the enzyme was prepared in which heme a/CuA are oxidized and heme a3/CuB are reduced with CO bound to heme a3, which stabilizes the reduced state of the binuclear center. In addition, in this state the pKs of protonatable groups in the vicinity of the binuclear center, interacting electrostatically with heme a3, are larger than with oxidized heme a3. Upon flash photolysis of CO from the two-electron reduced enzyme electrons at heme a3 equilibrate rapidly with heme a. In the R. sphaeroides enzyme the electron-transfer rates from heme a to a3 and from heme a3 to a were, deconvoluted and were found to be approximately 1.5.10(5) s-1 and approximately 1.4.10(5) s-1, respectively. After this rapid electron equilibration between hemes a and a3, protons are released from groups interacting electrostatically with heme a3, which is associated with additional electron transfer from heme a3 to heme a. The proton-coupled electron transfer displays a pH dependent extent and rate. In addition, it displays a deuterium-isotope effect of a factor of about three. The reaction sequence is compatible with the three-dimensional cytochrome c oxidase structure, which shows that more protonatable groups are found around heme a3 than around heme a and supports the involvement of the binuclear center in proton pumping. Proton uptake/release upon reduction/oxidation of heme a3 takes place through a proton pathway including residues Thr(I-359) and Lys(I-362) (K-pathway), but not through the pathway including residues Asp(I-132) and Glu(I-286) (D-pathway). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway.

Amino Acid Sequence↗
PubMed1997

Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen.

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.

Amino Acid Substitution↗
PubMed1997

Factors determining electron-transfer rates in cytochrome c oxidase: studies of the FQ(I-391) mutant of the Rhodobacter sphaeroides enzyme.

The mechanisms of internal electron transfer and oxygen reduction were investigated in cytochrome c oxidase from Rhodobacter sphaeroides (cytochrome aa3) using site-directed mutagenesis in combination with time-resolved optical absorption spectroscopy. Electron-transfer reactions in the absence of O2 were studied after flash photolysis of CO from the partly-reduced enzyme and the reaction of the fully-reduced enzyme with O2 was studied using the so-called flow-flash technique. Results from studies of the wild-type and mutant enzyme in which phenylalanine-391 of subunit I was replaced by glutamine (FQ(I-391)) were compared. The turnover activity of the mutant enzyme was approximately 2% ( approximately 30 s-1) of that of the wild-type enzyme. After flash photolysis of CO from the partly-reduced mutant enzyme approximately 80% of CuA was reduced, which is a much larger fraction than in the wild-type enzyme, and the rate of this electron transfer was 3.2 x 10(3) s-1, which is significantly slower than in the wild-type enzyme. The redox potentials of hemes a and a3 in the mutant enzyme were found to be shifted by about +30 and -70 mV, respectively, as compared to the wild-type enzyme. During the reaction of the fully-reduced FQ(I-391) mutant enzyme with O2 a rapid kinetic phase with a rate constant of 1.2 x 10(5) s-1, presumably associated with O2 binding, was followed by formation of the P intermediate with electrons from heme a3 and CuB with a rate of approximately 4 x 10(3) s-1, and oxidation of the enzyme with a rate of approximately 30 s-1. The dramatically slower electron transfer between the hemes during O2 reduction in the mutant enzyme is not only due to the slower intrinsic electron transfer, but also due to the altered redox potentials. In addition, the results show that the reduced overall activity of the mutant enzyme is due to the slower electron transfer from heme a to the binuclear center during O2 reduction. The relation between the intrinsic heme a/heme a3 electron-transfer rate and equilibrium constant, and the electron-transfer rate from heme a to the binuclear center during O2 reduction is discussed.

Electron Transport↗
PubMed1996

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.

Animals↗
PubMed1996

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.

Carbon Monoxide↗
PubMed1996

A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.

The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a3, as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a3, nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.

Base Sequence↗
PubMed1996

A flash-photolysis study of the reactions of a caa3-type cytochrome oxidase with dioxygen and carbon monoxide.

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase from Bacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochrome c subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 microseconds, 30 microseconds, 100 microseconds, and I ms at 445 nm. The initial, 10-microsecond absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving heme a and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochrome c, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from heme a3 to a in PS3 oxidase compared to the bovine enzyme.

Animals↗
PubMed1995

EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center.

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging carboxyl ligand in the bimetallic cytochrome a3-CuB site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the carboxyl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Base Sequence↗
PubMed1995

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+.

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Internal electron transfer in cytochrome c oxidase from Rhodobacter sphaeroides.

Absorbance changes following CO dissociation by flash photolysis from mixed-valence aa3 cytochrome oxidase from Rhodobacter sphaeroides have been followed in the Soret and alpha regions. They reflect internal electron transfer in the partially reduced enzyme, and the kinetics of the reactions has been determined. As with the bovine enzyme, three kinetic phases are found with relaxation time constants at neutral pH of about 3 microseconds, 35 microseconds, and 1 ms. The first reaction phase represents electron transfer from cytochrome a3 to cytochrome a, and the extent of this reaction is about 3 times larger compared to the bovine enzyme. The energetics of the reaction has been analyzed on the basis of measurements of its temperature dependence. The reorganization energy is close to 120 kJ mol-1, and it is suggested that this rather high value is the result of changes in solvation at the cytochrome a3-CuB site. The subsequent electron transfer between cytochrome a and CuA, with a time constant of 35 microseconds, is almost activationless and has a very low reorganization energy. The final phase, with a time constant close to 1 ms at neutral pH, represents a further shift in the equilibrium between cytochrome a3 and cytochrome a, and it is limited by proton-transfer reactions. The pKa values of the groups involved are significantly shifted in the bacterial oxidase compared to the bovine one. The total extent of electron transfer in the three backflow reactions has also been determined by a comparison of the CO-recombination rates in the mixed-valence and fully reduced enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Triplet-state quenching in complexes between Zn-cytochrome c and cytochrome oxidase or its CuA domain.

The quenching of the triplet state of Zn-cytochrome c in electrostatic complexes with cytochrome oxidase and its soluble CuA domain has been studied by laser flash photolysis. The triplet state of free Zn-cytochrome c decayed with a rate of about 200 s-1. With the oxidase, biphasic decay with rate constants of 2 x 10(5) and 2 x 10(3) s-1, respectively, was observed. At high ionic strength (I = 0.2) the decay was the same as with free Zn-cytochrome c. The quenching was also eliminated by reduction of the oxidase. The decay rate in the complex with the CuA domain was 4 x 10(4) s-1. The results are interpreted in terms of rapid electron transfer to CuA and a slower one to cytochrome a. No electron transfer products were detected, because the backward reaction is faster than the forward one. This can be explained by the high driving force (1.1 eV) for the forward electron transfer, taking the system into the inverted Marcus region. The distance in the electrostatic complex between cytochrome c and the electron acceptor, presumed to be CuA, is calculated to be 16 A.

Animals↗