Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m307594200
ISSN1083-351X
AutoresOliver Maneg, Bernd Ludwig, Francesco Malatesta,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoCytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brønsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases. Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brønsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases. The aerobic electron transport chain of Paracoccus denitrificans represents a model system for the mitochondrial respiratory chain, where the terminal reaction, the reduction of dioxygen to water, is mediated by cytochrome-c oxidase (EC 1.9.3.1). The electrons for this reaction are donated by a c-type cytochrome (7Baker S.C. Ferguson S.J. Ludwig B. Page M.D. Richter O.M.H. van Spanning R.J. Microbiol. Mol. Biol. Rev. 1998; 62: 1046-1078Crossref PubMed Google Scholar) and enter the oxidase via the CuA center (for a recent review see Ref. 2Schultz B.E. Chan S.I. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 23-65Crossref PubMed Scopus (200) Google Scholar), a highly conserved motif in subunit II of cytochrome-c oxidases of eukaryotes, aerobic bacteria, and in the nitrous oxide reductase of denitrifying bacteria. The CuA center resides in a periplasmic, solvent-exposed domain of subunit II and contains two copper ions in a mixed valence state, which give rise to the typical purple color of the isolated domain (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar). The two copper atoms are bound by two cysteine residues forming thiolate bridges, two histidine residues, and as further ligands a methionine sulfur and a glutamate peptide carbonyl. Soluble domains of several bacterial cytochrome-c oxidases have been prepared including P. denitrificans (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar), T. thermophilus (4Slutter C.E. Sanders D. Wittung P. Malmström B.G. Aasa R. Richards J.H. Gray H.B. Fee J.A. Biochemistry. 1996; 35: 3387-3395Crossref PubMed Scopus (125) Google Scholar), Paracoccus versutus (5Salgado J. Warmerdam G. Bubacco L. Canters G.W. Biochemistry. 1998; 37: 7378-7389Crossref PubMed Scopus (58) Google Scholar), and Bacillus subtilis (6von Wachenfeldt C. de Vries S. van der Oost J. FEBS Lett. 1994; 340: 109-113Crossref PubMed Scopus (100) Google Scholar). Following electron transfer to the CuA center from cytochrome c, electrons are further transferred to the low-spin heme a (or b) in subunit I, in a very fast μs time-scale process, and finally to the binuclear heme a3-CuB site (on a ms time scale), where dioxygen is reduced to water. In P. denitrificans two c-type cytochromes have been suggested to mediate the ET 1The abbreviations used are: ETelectron transferTEVtobacco etch virus. processes between the bc1 complex and the terminal electron accepting enzymes, the aa3- and the cbb3-type cytochrome-c oxidases, and the nitrite and the nitrous oxide reductases of the nitrate respiratory pathway. A soluble cytochrome c550 is believed to function as an electron donor in different respiratory pathways such as in methanol and methylamine oxidation or in denitrification (7Baker S.C. Ferguson S.J. Ludwig B. Page M.D. Richter O.M.H. van Spanning R.J. Microbiol. Mol. Biol. Rev. 1998; 62: 1046-1078Crossref PubMed Google Scholar). The kinetics of the electron transfer reaction between this cytochrome and a soluble CuA domain from P. denitrificans aa3 cytochrome-c oxidase have been studied (8Lappalainen P. Watmough N.J. Greenwood C. Saraste M. Biochemistry. 1995; 34: 5824-5830Crossref PubMed Scopus (77) Google Scholar). However, there is ample evidence that a different cytochrome, cytochrome c552, is the genuine mediator between the bc1 complex and aa3. This 18-kDa cytochrome (9Turba A. Jetzek M. Ludwig B. Eur. J. Biochem. 1995; 231: 259-265PubMed Google Scholar) is composed of three functional domains: an N-terminal helical membrane anchor, a negatively charged spacer region, and a typical class I c-type heme domain. This cytochrome is believed to be the bona fide electron transfer shuttle protein between the bc1 complex and aa3, because (i) a ternary supercomplex consisting of these three components is isolated under certain detergent solubilization conditions from P. denitrificans membranes (10Berry E.A. Trumpower B.L. J. Biol. Chem. 1985; 260: 2458-2467Abstract Full Text PDF PubMed Google Scholar); (ii) electron transport from NADH to dioxygen in isolated membranes is blocked in deletion mutants lacking the c552-coding gene, but this inhibition may be overcome by mitochondrial cytochrome c (11Turba A. Molekularbiologische und biochemische Charakterisierung des Membran-gebundenen Cytochrom c552 aus Paracoccus denitrificans. Ph.D. thesis, University of Frankfurt, Frankfurt, Germany1993Google Scholar), and (iii) specific antibodies directed against purified cytochrome c552 can block electron transport from NADH to oxygen in membrane activity assays (9Turba A. Jetzek M. Ludwig B. Eur. J. Biochem. 1995; 231: 259-265PubMed Google Scholar). electron transfer tobacco etch virus. At low ionic strength (i.e. below 10 mm), the reaction between mitochondrial cytochrome c and cytochrome-c oxidase involves the formation of a 1:1 stoichiometric complex, which may be isolated in vitro (12Dethmers J.K. Ferguson-Miller S. Margoliash E. J. Biol. Chem. 1979; 254: 11973-11981Abstract Full Text PDF PubMed Google Scholar). Under these conditions, complex formation is believed to rate-limit the subsequent rapid intracomplex ET process (13Antalis T.M. Palmer G. J. Biol. Chem. 1982; 257: 6194-6206Abstract Full Text PDF PubMed Google Scholar). From the strong ionic strength dependence of the reaction and from mutagenesis studies (14Witt H. Malatesta F. Nicoletti F. Brunori M. Ludwig B. Eur. J. Biochem. 1998; 251: 367-373Crossref PubMed Scopus (80) Google Scholar, 15Drosou V. Malatesta F. Ludwig B. Eur. J. Biochem. 2002; 269: 2980-2988Crossref PubMed Scopus (39) Google Scholar) a two-step model has been proposed for the interaction of the proteins. Initially the orientation is mediated by long range electrostatic forces, followed by the fine-tuning of the interaction by hydrophobic patches within the docking site. In contrast to this, nearly no charged residues are found on the probable interaction interfaces of the corresponding proteins from T. thermophilus, as demonstrated by the recently solved crystal structures of the ba3 cytochrome-c oxidase (16Soulimane T. Buse G. Bourenkov G.P. Bartunik H.D. Huber R. Than M.E. EMBO J. 2000; 19: 1766-1776Crossref PubMed Scopus (408) Google Scholar) and its substrate (17Than M.E. Hof P. Huber R. Bourenkov G.P. Bartunik H.D. Buse G. Soulimane T. J. Mol. Biol. 1997; 271: 629-644Crossref PubMed Scopus (79) Google Scholar), a soluble cytochrome spectroscopically identified as c552 (18Soulimane T. von Walter M. Hof P. Than M.E. Huber R. Buse G. Biochem. Biophys. Res. Commun. 1997; 237: 572-576Crossref PubMed Scopus (53) Google Scholar). T. thermophilus is a Gram-negative, extremely thermophilic eubacterium found in hot springs with optimum growth temperature around 75 °C. Taking into consideration that the stability of electrostatic interactions is lower at higher temperatures, hydrophobic interactions become more favorable. Kinetic studies have shown that the turnover activity of cytochrome c552 with ba3 oxidase becomes faster as ionic strength is decreased (19Giuffrè A. Forte E. Antonini G. D'Itri E. Brunori M. Soulimane T. Buse G. Biochemistry. 1999; 38: 1057-1065Crossref PubMed Scopus (73) Google Scholar). On the contrary, P. denitrificans aa3 cytochrome-c oxidase shows very low turnover rates under low ionic strength conditions, most likely because of the formation of a high affinity electrostatic complex (15Drosou V. Malatesta F. Ludwig B. Eur. J. Biochem. 2002; 269: 2980-2988Crossref PubMed Scopus (39) Google Scholar). Elucidation of the cytochrome c-CuA electron transfer mechanism is complicated by the subsequent monomolecular ET events taking place along the reaction coordinate that complete the reduction of dioxygen to water. It was, therefore, of interest to isolate and compare both periplasmically oriented subunit II CuA domains from P. denitrificans and T. thermophilus and to study the electron transfer reactions with their cytochrome c552 electron donor counterparts. Differences between the two systems with respect to the relevant electron transfer reactions can be studied directly by stopped-flow spectroscopy without interference from heme a absorbance and by the subsequent electron transfer and energy transduction events. The results indicate that the expressed soluble CuA domains are excellent models for the initial electron transfer events in cytochrome-c oxidases. Expression and Purification Procedures—Expression of the CuA fragment of P. denitrificans subunit II, encoded by the ctaC gene (20Steinrücke P. Steffens G.C. Panskus G. Buse G. Ludwig B. Eur. J. Biochem. 1987; 167: 431-439Crossref PubMed Scopus (67) Google Scholar), was carried out in Escherichia coli JM 109. The region representing amino acid residues 130–280 of the CuA domain was amplified by PCR. The subunit II-BamHI-TEV primer with sequence actgggatccgaaaacctatacttccaaagccaggagatgccgaacg (BamHI site underlined, TEV site in italics) was used as forward primer, coding for the TEV-protease recognition site (ENLYFQS, with cleavage between Q and S). As a reverse primer an oligonucleotide was used according to Lappalainen et al. (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar), introducing a HindIII site, to allow for cloning into the expression vector pQE-30 (Qiagen). This construct provides considerably higher expression rates (up to 8 mg/liter) than the previous method (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar). Because the His tag (coded on the parent vector) is not used for purification, nor is its presence in the protein desirable, it was cleaved off in vivo by co-transforming pRK603 into this E. coli strain (21Kapust R.B. Waugh D.S. Protein Expr. Purif. 2000; 19: 312-318Crossref PubMed Scopus (164) Google Scholar), thus providing constitutive expression of the TEV-protease. Cells were grown on minimal medium containing 40 g/liter glycerol, 7.5 g/liter K2HPO4, 5.3 g/liter NaH2PO4-H20, 2 g/liter NH4Cl, 1 g/liter glucose, 1 mm MgSO4, 0.1 mm CaCl2, 10 ml/liter trace element solution 1 (22Wingfield P.T. Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, Inc., New York1998Google Scholar), 0.2 μm thiamin, 100 μg/ml ampicillin, 25 μg/ml kanamycin, in a 10-liter New Brunswick Microferm fermentor. Cells were induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at an A600 of ∼4.0. After 4 h cells were harvested, and preparation of inclusion bodies, protein refolding, copper insertion, and purification were carried out essentially as described previously (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar). Gel filtration was done on Sephacryl HR S200 (Amersham Biosciences), and an additional purification step on a nickel-nitrilotriacetic acid column (Qiagen) in 20 mm Bis-Tris buffer at pH 7.0 removed residual His-tagged material, as determined by SDS-PAGE (not shown). The CuA fragment of the T. thermophilus ba3 cytochrome-c oxidase was expressed and purified as described previously (4Slutter C.E. Sanders D. Wittung P. Malmström B.G. Aasa R. Richards J.H. Gray H.B. Fee J.A. Biochemistry. 1996; 35: 3387-3395Crossref PubMed Scopus (125) Google Scholar). The cytochrome c552 soluble domain of P. denitrificans was expressed in E. coli according to the published protocol (23Reincke B. Thöny-Meyer L. Dannehl C. Odenwald A. Aidim M. Witt H. Rüterjans H. Ludwig B. Biochim. Biophys. Acta. 1999; 1411: 114-120Crossref PubMed Scopus (51) Google Scholar). Following a similar approach, the T. thermophilus cytochrome c552 gene was cloned into pET22b, a vector providing the pelB leader, which directs the protein to the periplasm. Correct assembly and insertion of the cofactor into the apoprotein was achieved by cotransforming E. coli cells with the heme maturation plasmid pEC86 (24Arslan E. Schulz H. Zufferey R. Künzler P. Thöny-Meyer L. Biochem. Biophys. Res. Commun. 1998; 251: 744-747Crossref PubMed Scopus (341) Google Scholar). Purification was carried out according to Fee et al. (25Fee J.A. Chen Y. Todaro T.R. Bren K.L. Patel K.M. Hill M.G. Gomez-Moran E. Loehr T.M. Ai J. Thöny-Meyer L. Williams P.A. Stura E. Sridhar V. McRee D.E. Protein Sci. 2000; 9: 2074-2084Crossref PubMed Scopus (52) Google Scholar). Protein concentration was determined by using the following extinction coefficients: Pd-c552 ΔϵRed-Ox, 552 nm = 19.4 mm–1 cm–1, Pd-CuA ϵ480 nm = 3.0 mm–1 cm–1, Tt-c552 ΔϵRed-Ox, 552 nm = 21.0 mm–1 cm–1, Tt-CuA ϵ530 nm = 3.1 mm–1 cm–1. Stopped-flow Spectroscopy and Experimental Protocol—Kinetic experiments were carried out by using a thermostatted Applied Photophysics stopped-flow apparatus (Leatherhead, United Kingdom) with a 1-cm observation chamber. The ET kinetics between the soluble CuA domains and cytochromes c were studied in both the forward (physiological) and reverse directions according to the following experimental protocol (Scheme 1), which was devised to (i) minimize the auto-oxidizability of the reduced proteins, and to (ii) better control the initial absolute concentration of the reduced proteins. According to Scheme 1, Scheme I one of the partner proteins is initially reduced anaerobically in the stopped-flow syringe by ascorbate (see vertical arrows), which at the chosen solution pH is a slow reductant (see below). Following complete reduction, the solution is mixed in the stopped-flow apparatus with an anaerobic solution containing the oxidized ET acceptor protein, and the time-dependent extinction changes followed at 551 nm (P. denitrificans) or at 552 nm (T. thermophilus). Thus, in the forward direction cytochrome c is reduced by ascorbate and mixed with oxidized CuA, whereas in the reverse direction prereduced CuA is mixed with oxidized cytochrome c. In all experiments the buffer (20 mm Bis-Tris, pH 7.0, with ionic strength varied by appropriate amounts of KCl) was flushed with N2 in a glass gas-tight syringe (fitting the stopped-flow valve) for at least 15 min, and following addition of the protein of interest, the solution was flushed for an additional 15 min. Finally 0.5 mm sodium ascorbate was added from a 1.0 m stock solution. All experiments were performed at 8 °C. Complete reduction was achieved after several minutes, as determined separately in stopped-flow experiments in which sodium ascorbate, at varying pseudo-first order concentrations, was mixed with either oxidized protein (see Fig. 1 and “Results and Discussion”). The apparent bimolecular rate constants for reduction of the CuA domains both from P. denitrificans and T. thermophilus and the corresponding soluble cytochromes c552 were in the range of 60 to 330 m–1 s–1 (results not shown), 3–5 orders of magnitude smaller than the ET process of interest. Under all the experimental conditions tested (cytochrome c concentration, ionic strength, and temperature), the forward and reverse ET apparent bimolecular rate constants were determined by fitting the observed kinetic traces to a simple exponential relaxation process at different ferro- or ferricytochrome c concentrations (forward and reverse directions, respectively) followed by linear regression of the observed rate constants to the varied cytochrome c concentrations. Three or more kinetic traces were acquired for each specific experimental condition and averaged. True pseudo-first order conditions could not be completely achieved throughout the kinetic titration experiments because of the high cytochrome c (19.4–21.0 mm–1 cm–1) and low CuA (3–3.1 mm–1 cm–1) extinction coefficients on one hand, and to the relatively high reaction rates observed especially at low ionic strength for the P. denitrificans couple, which approached the time resolution of the stopped-flow apparatus (dead time of 1.3 ms, determined by using the myoglobin-carbon monoxide combination reaction) on the other. Apparent second-order rate constants were determined from the slopes of the linear portions of the pseudo-first order plots (see “Results and Discussion”). Data fitting was performed by using either the Matlab (The MathWorks Inc.) or Scientist (Micromath Scientific Software Inc.) softwares. The standard deviation of the fitted parameters never exceeded 10%. In the present investigation the kinetics of electron transfer between two genetically engineered ET couples from a mesophilic and a thermophilic species have been studied by stopped-flow spectroscopy. The experimental rationale has been to pre-reduce one protein of each ET couple with the kinetically sluggish reductant ascorbate and to subsequently mix the proteins and follow the interprotein ET events at a suitable wavelength (see “Experimental Procedures”). At pH 7 the concentration of the true reductant, i.e. the ascorbate dianion, is very low (26Al Ayash A.I. Wilson M.T. Biochem. J. 1979; 177: 641-648Crossref PubMed Scopus (52) Google Scholar, 27Myer Y.P. Thallam K.K. Pande A. J. Biol. Chem. 1980; 255: 9666-9673Abstract Full Text PDF PubMed Google Scholar) and therefore not expected to interfere with the ET events taking place between the partners of interest. Fig. 1 depicts a typical example of the Paracoccus couple, studied in the forward, physiological direction (bottom panel, mixing of fully reduced Pd-c552 with oxidized Pd-CuA) and in the reverse direction (top panel, in which oxidized Pd-c552 is mixed with reduced Pd-CuA) followed at 551 nm, where the extinction of Pd-c552 is dominant. In either direction, absorbance changes take place on a short time scale (0.2 and 0.1 s) indicating partial oxidation (bottom panel) or reduction (top panel) of Pd-c552. These experiments were carried out by varying the ascorbate concentration from 0.25 to 1 mm to exclude any competition of the reductant with the interprotein ET reaction of interest. Indeed as ascorbate concentration is increased, no significant change in rate or amplitude is observed on the short time scale, and all time courses display a simple exponential behavior. On longer time scales (50 s), however, the time-dependent absorbance changes appear to be linearly correlated to ascorbate concentration (not shown). These observations suggest that in the fast phase (Fig. 1, left part of top and bottom panels) interprotein ET occurs, taking the proteins to a transient equilibrium state controlled by the protein-chemical structural details and driven by the redox potential of the two ET proteins. On longer time scales (Fig. 1, right part of top and bottom panels) the ascorbate reaction takes the partially oxidized equilibrium ET couple mixture to the fully reduced state as expected from the larger driving force of ascorbate. Similar results were obtained with the Thermus thermophilus ET couple (not shown). We have systematically studied the cytochrome c552 concentration dependence of the interprotein ET reaction with both couples as a function of ionic strength, using the ascorbate (0.25 mm after mixing) protocol described above. All observed fast phases could be fitted to a single exponential time course. The results of these experiments are shown in Figs. 2 and 3 (P. denitrificans couple) and Fig. 4 (T. thermophilus couple) in which the observed fitted rate constants are plotted as a function of cytochrome c552 concentration. The pseudo-first order plots were linear with respect to the varied cytochrome c552 concentration, although, especially at low cytochrome concentrations, there was some deviation from linear behavior. This is expected, because at low cytochrome c552 concentrations the reaction mixture is not under true pseudo-first order conditions. Nevertheless from the slope of the linear portion of the plots the apparent second-order rates for the interprotein ET reaction could be estimated and are given in Tables I and II for the P. denitrificans and T. thermophilus couple, respectively, at different ionic strength values. As can be seen from the reported data, the ET reaction of the P. denitrificans couple is strongly dependent on ionic strength with second-order rate constants ranging from ∼4*106 to 9*104m–1 s–1 and from 2*107 to 6*105m–1 s–1, for the forward and reverse ET reactions, respectively, as ionic strength is increased from 10 to 200 mm (see Table I). The apparent equilibrium constant for the physiological direction is thus about 0.2 for the P. denitrificans couple and does not appear to vary significantly with ionic strength. Although this result indicates a higher stability of reduced Pd-c552 over oxidized Pd-CuA, (i) this has also been observed previously (28Szundi I. Cappuccio J.A. Borovok N. Kotlyar A.B. Einarsdottir O. Biochemistry. 2001; 40: 2186-2193Crossref PubMed Scopus (29) Google Scholar) in the reaction between bovine heart cytochrome-c oxidase and yeast iso-1-cytochrome c, supporting our result; (ii) the reported redox potentials of the P. denitrificans couple are 270 and 240 mV for Pd-c552 and Pd-CuA, respectively (3Lappalainen P. Aasa R. Malmström B.G. Saraste M. J. Biol. Chem. 1993; 268: 26416-26421Abstract Full Text PDF PubMed Google Scholar, 29Schneider M. Zielgerichtete Mutagenese am Cytochrom C552 aus Paracoccus denitrificans. Diploma thesis, University of Frankfurt, Frankfurt, Germany2000Google Scholar), which yields an equilibrium constant of about 0.3, in close agreement with our findings; and (iii) the ensuing exergonic ET reactions to the low spin heme a and the a3-CuB center that account for O2 reduction drive the thermodynamically unfavorable reaction in the physiological direction.Fig. 3The reverse electron transfer reaction between Paracoccus cytochrome c552 and the CuA fragment. The fast phase fitted rate constants of the ET reaction (see text and Fig. 1, top panel) between ferric Pd-c552 and reduced Pd-CuA (with concentrations in the range from 8.1 to 9.8 μm after mixing) at various ionic strength conditions (15 mm, ▴; 25 mm, ▾; 35 mm, ♦; 50 mm, +; 100 mm, ×; 200 mm, ▪) are plotted against the initial ferric Pd-c552 concentration. Ascorbate concentration is 0.25 mm after mixing. Solid lines were determined by linear regression of the data points. Other conditions were as in Fig. 1. T = 8 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The electron transfer reaction between Thermus cytochrome c552 and the CuA fragment. The fast phase fitted rate constants of the ET reaction between the Tt-c552 and Tt-CuA (forward direction, with concentrations in the range from 8.7 to 9.3 μm after mixing; reverse direction, 10.6 μm after mixing) couple are plotted as a function of the initial ferro- or ferri-Tt-c552 concentration. The forward direction was studied at the following ionic strength values: 15 mm (□); 25 mm (•); 50 mm (▴); and 100 mm (▾). The reverse reaction was only studied at an ionic strength of 25 mm (♦; dotted line). Lines were obtained by linear regression of the data points. Ascorbate concentration is 0.25 mm after mixing. Observation wavelength, 552 nm. Other conditions were as in Fig. 1. T = 8 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IApparent second-order rate constants for the Paracoccus ET couple N.D., not determined.Ionic strengthkforwardkreversekforward/kreversemmm-1s-1104.13·106N.D.N.D.153.42·10617.7·1060.19252.05·10610.6·1060.19351.46·1068.1·1060.18500.83·1065.0·1060.171000.36·1061.9·1060.192000.09·1060.6·1060.15 Open table in a new tab Table IIApparent second-order rate constants for the Thermus ET couple N.D., not determined.Ionic strengthkforwardkreversekforward/kreversemmm-1s-1153.50·106N.D.N.D.253.35·1060.7·1064.8503.27·106N.D.N.D.1002.62·106N.D.N.D. Open table in a new tab In contrast to these findings the ionic strength dependence of the T. thermophilus ET couple is very modest with rates in excess of 106m–1 s–1 in either direction at the lowest ionic strength (see Fig. 4 and Table II). From these data an equilibrium constant of 4.8 was calculated for the Thermus protein pair, indicating that in this case the physiological direction is thermodynamically favored for the isolated domains. Several values of the redox potentials, depending strongly on the experimental conditions (temperature, ionic strength, pH), of the Thermus proteins have been reported (Th-CuA 240 mV up to 266 mV; see Refs. 4Slutter C.E. Sanders D. Wittung P. Malmström B.G. Aasa R. Richards J.H. Gray H.B. Fee J.A. Biochemistry. 1996; 35: 3387-3395Crossref PubMed Scopus (125) Google Scholar and 30Inmoos C. Hill M.G. Sanders D. Fee J.A. Slutter C.E. Richards J.H. Gray H.B. J. Biol. Inorg. Chem. 1996; 1: 529-531Crossref Scopus (46) Google Scholar; Tt-c552 200 mV (25Fee J.A. Chen Y. Todaro T.R. Bren K.L. Patel K.M. Hill M.G. Gomez-Moran E. Loehr T.M. Ai J. Thöny-Meyer L. Williams P.A. Stura E. Sridhar V. McRee D.E. Protein Sci. 2000; 9: 2074-2084Crossref PubMed Scopus (52) Google Scholar) and 230 mV (31Hon-Nami K. Oshima T. J. Biochem. (Tokyo). 1977; 82: 769-776Crossref PubMed Scopus (54) Google Scholar)), complicating the analysis and the comparison with the reported equilibrium constant gained from the kinetic data. However, calculation of the equilibrium constant with any of these values always confirms the physiological direction to be favored. From the data shown in Figs. 2, 3, 4, as well as Tables I and II, Fig. 5 was constructed in which the logarithm of the apparent bimolecular rate constant is plotted as a function of the square root of ionic strength of the solution. As predicted by the Brønsted law (32Brønsted J.N. La Mer V.K. J. Am. Chem. Soc. 1924; 46: 555-573Crossref Scopus (20) Google Scholar), shown below in Equation 1, where k is the observed bimolecular rate constant at ionic strength I, k0 the bimolecular rate constant at I = 0, B a term whose value, ∼0.5 at 8 °C, is derived from Debye-Hückel eq
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