Tài liệu Báo cáo Y học: Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase Electron entry and kinetic phases of the reaction pptx

Mutations in the docking site for cytochrome c on the Paracoccus heme aa 3 oxidase Electron entry and kinetic phases of the reaction Viktoria Drosou 1 , Francesco Malatesta 2 and Bernd Ludwig 1 1 Molecular Genetics, Institute of Biochemistry, Johann-Wolfgang-Goethe Universita ¨ t, Frankfurt, Germany; 2 Department of Basic and Applied Biology, University of L’Aquila, Italy Introducing site-directed mutations in surface-exposed residues of subunit II of the heme aa 3 cytochrome c oxidase of Paracoccus denitrificans, we analyze the kinetic para- meters of electron transfer from reduced horse heart cyto- chrome c. Specifically we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics. From our data we conclude that tryptophan 121 on subunit II is the sole entry point for electrons on their way to the Cu A center and that its precise spatial arrangement, but not its aromatic nature, is a prerequisite for efficient electron transfer. With different reaction partners and experimental conditions, biphasicity can always be induced and is critically dependent on the ionic strength during the reaction. For an alternative explanation to account for this phenomenon, we find no evidence for a second cytochrome c binding site on oxidase. Keywords: Paracoccus denitrificans; cytochrome c oxidase; docking site; electron transfer; biphasic kinetics. Cytochrome c oxidase is the terminal complex of the respiratory chains of mitochondria and many bacteria [1–4]. It catalyzes the reduction of oxygen to water, coupling the free energy of this reaction to the generation of a proton gradient across the membrane. During the redox reaction, an electron delivered from cytochrome c is first transferred to Cu A , a binuclear copper center located close to the surface of the large hydrophilic domain of subunit II. It is then donated to heme a embedded in subunit I, and subsequently to the heme a 3 Æ Cu B center where oxygen reduction, and most likely the redox coupling to proton pumping, take place. While the mitochondrial enzyme comprises up to 13 different subunits in a dimeric complex, the oxidase of the bacterium Paracoccus denitrificans consists of only four subunits, with the three largest ones homologous to the corresponding mitochondrial subunits. Typically, many isolated oxidases are somewhat promis- cuous towards their substrate molecules. Early studies analyzing the surface properties of cytochromes c of different origin revealed a basic cluster of mostly lysine residues located around the heme crevice. Being responsible for docking to their redox partners, an interaction between cytochrome c and oxidase based on electrostatic forces was described [5–7]. Experiments with monoclonal antibodies directed against subunit II of cytochrome c oxidaseleadtoa loss of activity [8] and supported the notion that the catalytic binding site is located predominantly on subunit II. This result was confirmed by chemical modifications and early site-directed mutagenesis experiments [9,10], and is consis- tent with the crystal structures of the eukaryotic and the Paracoccus denitrificans oxidase showing clusters of negat- ively charged residues on the surface of subunit II [11,12]. Previous studies on the binding of cytochrome c to the Paracoccus oxidase were interpreted by a two-step model in which electrostatic forces are responsible for an efficient long-range docking, followed by the reorientation of the redox partner driven by hydrophobic interactions [13]. Specifically, a set of four acidic residues exposed on subunit II (D135, D178, and to a lesser extent, E126, D159; see Fig. 1 and [13]) had been assumed to interact electrostat- ically with the horse heart cytochrome c. While a pivotal role in electron transfer from cytochrome c was assigned to residue W121 on subunit II [14], this early study did not address the question whether any other (aromatic) side chains in this or the neighbouring position might be able to support electron transfer to the Cu A site, thereby function- ally replacing tryptophan 121. As already observed previously, oxidase kinetics may yield nonlinear Eadie–Hofstee plots (e.g. [15]). Two different phases, denoted high and low affinity, are clearly discern- ible, each being characterized by a set of individual kinetic parameters. These biphasic steady-state kinetics become monophasic at higher ionic strength, a phenomenon discussed in terms of different binding sites (e.g. [15,16]). or of conformational changes within the enzyme-substrate complex related to the coupling of electron transfer with proton pumping [17]. Correspondence to B. Ludwig, Molecular Genetics, Institute of Biochemistry, Biozentrum, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany. Fax: + 49 69 798 29244, Tel.: + 49 69 798 29237, E-mail: ludwig@em.uni-frankfurt.de Abbreviations:I,ionicstrength;c 552 -f: soluble fragment of the Paracoccus denitrificans cytochrome c 552 , expressed and purified from Escherichia coli. (Received 4 February 2002, revised 12 April 2002, accepted 2 May 2002) Eur. J. Biochem. 269, 2980–2988 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02979.x Here we give a comprehensive description of the inter- action domain for cytochrome c on subunit II of the P. denitrificans oxidase, specifically addressing (a) the electron entry point to oxidase and its possible alternatives, (b) the size and extent of the acidic patch on subunit II, and (c) ways of experimentally influencing the kinetic phases during enzyme turnover. MATERIALS AND METHODS Mutagenesis and enzyme preparation Site-directed mutagenesis in the ctaCgenewasper- formed according to the Ôaltered siteÕ mutagenesis protocol (Promega, Heidelberg). Complementation of the oxidase- deficient deletion mutant ST4 was performed as described previously [18]. Mutant strains were grown aerobically in succinate medium [19] including streptomycin sulfate (25 lgÆmL )1 ), membranes isolated according to [20] and solubilized using n-dodecyl b- D -maltoside. The four-subunit cytochrome c oxidase was purified by conventional chromatographic protocol as described in [21]; the oxidase complexed with an antibody fragment (F v )was isolated in a single chromatographic step as described previously [22,23], and excess F v removed by gel filtration. The two-subunit oxidase complex was prepared by combining the standard purification in dodecyl maltoside [21], however, running the gel filtration step in the presence of Triton X-100 to dissociate subunits III and IV [24]. Briefly, the following steps were performed: The superna- tant after ultracentrifugation was loaded on the first column, a DEAE-Sepharose CL-6B (Pharmacia Biotech) equilibrated with 20 m M potassium phosphate pH 8.0, 1m M EDTA and 0.5 gÆL )1 dodecyl maltoside. For elution, a linear gradient ranging from 100 to 600 m M NaCl was used with this buffer. Fractions of highest heme/protein ratio were pooled and concentrated in an Amicon cell (cut off 30 kDa). Triton X-100 was added to a final concentra- tion of 10% (w/v). The solution was stirred for 1 h at 4 °C, applied to an Ultrogel AcA34 (IBF Biotechniques) gel filtration column equilibrated and processed with the above buffer with 2 gÆL )1 Triton X-100 replacing the dodecyl maltoside. To reintroduce this latter detergent for subse- quent steps (and to avoid the detrimental effects of Triton X-100 on activity [25]), pooled fractions were rechromato- graphed on the first column equilibrated in 0.2 gÆL )1 dodecyl maltoside, and eluted with a linear gradient from 100 to 400 m M NaCl. The oxidase fractions were analyzed by SDS/PAGE to verify their subunit composition, con- centrated and stored at )80 °C. Steady-state kinetics and determination of the ionic strength dependence Cytochrome oxidase activity was measured with a Kontron Uvikon 941 spectrophotometer at 25 °Cin20m M Tris/ HCl, pH 7.5, 1 m M EDTA, 0.2 gÆL )1 n-dodecyl b- D -malto- side. The different ionic strength conditions were adjusted by adding KCl. Ferrocytochrome c (horse heart, Sigma) was prepared by reduction with dithionite and excess reductant removed by Sephadex G25 chromatography. Concentrations were varied between 0.5 and 40 l M ,and oxidation followed at 550 nm after adding the purified enzyme (40 p M up to 400 p M ). Determination the ionic- Fig. 1. The presumed docking site for cyto- chrome c on subunit II of the P. de nitrificans oxidase. The periplasmically oriented hydro- philic domain housing the homodimeric Cu A site (blue spheres) is depicted, omitting most of the two transmembrane helices (bottom). Selected side chains shown in detail were mutated; the residue crucial for electron entry, W121, is highlighted in yellow, along with other residues important for docking. The figure was prepared on the basis of the pub- lished coordinates (pdb1ar1), using the SWISS PDB VIEWER/POV RAY program [31]. Ó FEBS 2002 Cytochrome c docking site (Eur. J. Biochem. 269) 2981 strength (I) dependence was performed in the same buffer at 20 l M cytochrome c, with the ionic strength adjusted to values between 1.8 m M and 296 m M by the addition of KCl. The buffer for I ¼ 1.8 m M was 2.5 m M Tris/HCl, pH 7.5, 0.2 gÆL )1 dodecyl maltoside. Stopped-flow kinetics The presteady-state kinetics were followed on a ther- mostated Applied Photophysics DX.117 MV stopped- flow apparatus at 20 °Cin20m M potassium phosphate pH 7.6, 1 m M EDTA, 0.2 g L )1 dodecyl maltoside. The ionic strength was 140 m M , adjusted with KCl. Cytochrome oxidase (4–6 l M ) was incubated with 5 m M KCN at 4 °C for at least 6 h. Cytochrome c concentrations were varied in the range of 2–32 l M ; after mixing, the reaction was followed at 550 nm (oxidation of cytochrome c) and/or 605 nm (reduction of heme a), and biphasic time courses were obtained. Three independent measurements were done for each donor concentration, and the average was fitted to the sum of two exponentials. The observed rate constant from the fast phase of this double-exponential decay was plotted against the cytochrome c concentration, and the apparent bimolecular rate constant k on calculated from the slope. RESULTS The large, periplasmically oriented hydrophilic domain of subunit II of the heme aa 3 oxidase represents the major interaction site for cytochrome c. We constructed a set of mutants in surface-exposed residues to further identify amino acid side chains involved in the docking of cytochrome c, or in the presumed electron entry from cytochrome c to the Cu A center. Positions subjected to mutagenesis in this and two previous studies [13,14] are summarized in Fig. 1. All mutant enzymes, and several preparations of oxidase differing in polypeptide composi- tion, were assayed for their kinetic parameters under different ionic strength conditions. Ionic strength dependence of the turnover number The reaction of cytochrome c oxidase shows a strict dependence of the turnover number on ionic strength. We measured the oxidation of 20 l M horse heart cytochrome c by the various oxidase preparations under steady-state conditions, all yielding bell-shaped curves. While the optimum ionic strength for the wild-type enzyme was found to be 56 m M (Fig. 2; see also [13]), those for the two-subunit enzyme (Fig. 2) and all mutants in positions 121 and 122 (W121F, W121Y, W121Q/Y122Q, W121F/Y122F, W121G and W121Y/Y122W) were decreased to 36 m M .Ofthe remaining mutants, two showed wild-type behaviour (H119N, N160D), whereas all others were shifted to 46 m M (data not shown). Turnover and presteady-state kinetics We measured steady-state kinetics for all the subunit II mutants at their optimum ionic strength as determined above, using the reduced horse heart cytochrome c.The 46 m M ionic strength group was assayed both at 36 and at 56 m M , to allow for an unequivocal assignment to either a hyperbolic or nonhyperbolic Michaelis–Menten kinetic regime. Tables 1 and 2 list the relevant parameters, K m and k cat , for the different ionic strength conditions: the K m value is taken from the so-called high-affinity phase, and the k cat value is derived from the low-affinity phase. Positions W121 and Y122. A comprehensive set of single mutants in each of the two positions, or of double mutants, was generated (Table 1). While K m values for all complexes do not deviate from that of wild-type by more than a factor of 1.6, the catalytic activity of any mutant in the W121 position is drastically diminished. Residual activities for the two nonaromatic replacements (Q, G) are between 1 and 2%. Additional single mutations in the 121 position which exchange the tryptophan for two other aromatic residues (F, Y) show almost the same distinct loss of electron transfer activity, with residual k cat values around 3–5% compared to wild-type. When the neighbouring Y122 residue is changed to a phenylalanine, the kinetic behaviour is that of wild- type, and a glutamine in this position only reduces activity to 50%. We conclude that Y122 is not involved in the Fig. 2. Ionic strength dependence of the turnover number for the isolated four- and two-subunit Paracoccus heme aa 3 oxidase complex. The spectrophotometric assay was performed under steady-state condi- tions with 20 l M horse heart cytochrome c;forfurtherdetails,see Materials and methods. 4 su, four-subunit; 2 su, two-subunit complex. Table 1. Steady-state and stopped-flow parameters of horse heart cytochrome c oxidation by Paracoccus oxidase mutated in selected exposed aromatic residues of subunit II. The K m value was taken from the high-affinity phase at I ¼ 36 m M .Thek cat value was taken from the low-affinity phase at I ¼ 36 m M . NR, no rate measurable. Mutant position K m (l M ) k cat (s )1 ) k on · 10 6 ( M )1 Æs )1 ) Wild-type oxidase 1.4 669 3.7 W121Q 1.9 11 NR W121G 1.1 6 NR W121F 1.5 31 0.05 W121Y 1.3 22 0.03 Y122Q 2.2 333 0.4 Y122F 1.2 660 4.6 W121Q/Y122Q 1.7 9 NR W121Y/Y122W 1.2 7 0.13 W121F/Y122F 1.5 7 NR Y226F 1.3 626 2.4 2982 V. Drosou et al.(Eur. J. Biochem. 269) Ó FEBS 2002 electron transfer from cytochrome c to any large extent, nor is this position involved in maintaining the low residual activity when the W121 residue is mutated. Double mutants in both positions are not further diminished in activity compared to W121 single mutations (see Table 1). Further mutations (Y226F, H119N) in residues previously consid- ered as potential alternative entry points for electrons from cytochrome c (see Discussion) showed no deviations from wild-type in their kinetic properties. To exclude the possibility that diminished electron transfer activities in turnover experiments might be due to changes in redox properties of the first acceptor in oxidase, Cu A , we measured relevant redox steps in the W121F mutant, confirming that the redox potential for Cu A is in the wild-type range (P. Hellwig, Institut fu ¨ r Biophysik, Johann- Wolfgang, Goethe Universita ¨ t, Frankfurt, Germany, personal communication). Focussing on the parameters in Table 2 we found an increase of K m for mutants H119I/Q120I, D146N, E140Q and P196G measured at 56 m M . Comparing these values with those measured at 36 m M , again we found increased K m values for these mutants and also for E142Q. k cat as the parameter for maximum turnover is decreased. Mutants H119N and N160D reveal wild-type values; these positions do not seem to be involved in cytochrome c binding. We also assayed the oxidase mutants under presteady- state conditions, to ensure that the observed effects indeed relate to the early phases of electron entry. Using the cyanide-inhibited enzyme, the reaction sequence is limited to the transfer of the first two electrons reaching the Cu A /heme a redox couple. To shift cytochrome c oxidation kinetics into the time resolution of a stopped-flow apparatus, the reaction was followed at 140 m M ionic strength (see Materials and methods) and recorded at 550 nm (oxidation of cytochrome c) and at 605 nm (reduction of heme a). The observed time course was described by a sum of two exponentials. The fitted pseudo-first order rate constant was plotted against the cytochrome c concentration after mix- ing. From the slope of this linear plot the bimolecular rate constants k on were calculated for the wild-type and mutant enzymes (Tables 1 and 2). This analysis reflects and con- firms the k cat values obtained from turnover experiments. Some of the mutants showed extremely slow reduction behaviour, and bimolecular rates could not be determined (see Table 1). The k on values for the mutants Y122F, Y226F, N160D, H119N and H119I/Q120I are in the same range as the wild-type oxidase while the other mutants show a significantly decreased k on value (see Tables 1 and 2). Kinetic differences between the two-subunit and the four-subunit wild-type and mutant complexes To assess kinetic properties of both forms under identical detergent conditions, we prepared cytochrome c oxidase lacking both subunits III and IV by replacing the detergent in one of the chromatographic steps of the standard purification procedure (see Materials and methods): prior to gel filtration, the partially purified material was incubated with a large excess of Triton X-100, known to dissociate the oxidase and leave an enzymatically active two-subunit complex [24]. After gel filtration in Triton, dodecyl malto- side was reintroduced in the final step of column purification to exclude known detergent effects in the subsequent analysis [25]. Ionic strength dependency of the maximum turnover number was shifted from 56 m M for the four-subunit complex to 36 m M for the two-subunit preparation. Figure 2 also demonstrates that both complexes display a basically similar line shape, and turnover numbers are in close agreement at 20 l M cytochrome c. This behaviour is taken as a first evidence that the periplasmically oriented regions of one or both of the two ancillary subunits may contribute to some extent to the interaction domain for the substrate (see also Discussion). Kinetic parameters for both complexes under several ionic strength conditions are listed in Table 3. Comparing K m and k cat each at optimum ionic strength for both forms, it is evident that k cat is lower by a factor of three for the two- subunit enzyme, while its K m is diminished twofold. The overall specificity constant (k cat /K m ) of this two-subunit complex therefore remains in the same range, explaining in part its comparable activity at a given substrate concentra- Table 2. Oxidation of horse heart cytochrome c by wild-type oxidase and subunit II mutants under turnover and pre-steady state conditions at different ionic strengths. TM1, triple mutant (E126Q, D135N, D178N) in subunit II. ND, not determined. I ¼ 36 m M a I ¼ 56 m M I ¼ 140 m M Mutation K m (l M ) k cat (s )1 ) K m (l M ) k cat (s )1 ) k on · 10 6 ( M )1 Æs )1 ) b Wild-type oxidase 1.4 669 5.9 1031 3.7 E142Q 3.2 270 4.7 588 1.1 D146N 2.7 239 10.2 357 2.7 E140Q 5.6 277 7.0 250 2.1 D135N c 2.1 167 12.1 104 0.3 D178N c 2.0 213 15.0 313 2.3 TM1 c 7.9 25 ND ND ND P196G 1.1 345 10.4 714 1.5 H119I/Q120I ND ND 9.8 303 2.6 H119N ND ND 5.1 896 4.3 N160D ND ND 6.4 909 2.8 a Mutants as published in [13] were re-analyzed side by side, and are presented for a complete overview. b K m value taken from the high- affinity and k cat from the low-affinity phase. c Pre-steady-state kinetics of cytochrome c oxidation measured by stopped-flow, see Materials and methods for details. Ó FEBS 2002 Cytochrome c docking site (Eur. J. Biochem. 269) 2983 tion (Fig. 2). Analyzing lower ionic strength datasets for both preparations, the general trend persists that k cat is below that of the four-subunit enzyme, while K m values approach each other (see Table 3). Shifts from mono- to biphasic behaviour are observed for both the four-subunit and the two-subunit oxidase on going from higher ionic strength to lower values. Figure 3 exemplifies this transition to nonlinear kinetic behaviour for the two-subunit complex when [I] is diminished in steps from 56 to 15 m M . Eadie–Hofstee plots yield clear breaks for the two lower salt conditions (Fig. 3B). These transition points are listed in Table 3 (last column) for selected preparations/mutants (see also below). Also this criterion distinguishes the two-subunit variant from the four-subunit complex, where the transition occurs already at 36 m M , clearly indicating that biphasic kinetics are not due to the presence of subunits III and IV. Biphasic behaviour of the triple mutant TM1 (subunit II: E126Q, D135N, D178N) is evident when the four-subunit complex is assayed: while other mutants containing single acidic residue replacements followed biphasic kinetics at I ¼ 36 m M (not detailed), TM1 was monophasic at I ¼ 36 m M . Nevertheless, on further decreasing ionic strength, biphasic kinetics were again observed with a transition point at around 15 m M (see Table 3). The same holds true when the TM1 preparation was stripped of its subunits III and IV: the resulting two-subunit mutant complex displayed biphasic kinetics at 15 m M ionic strength. Further criteria for manipulating the kinetic phases of reaction Transitions from mono- to biphasic reaction conditions, depending on ionic strength variation, can be induced by other means as well. Specific F v fragments, derived from Table 3. Kinetic parameters and biphasic transitions under different ionic strength conditions for selected oxidase preparations. ND, not determined. I ¼ 7.4 m M I ¼ 14.8 m M I ¼ 26 m M I ¼ 36 m M I ¼ 56 m M Biphasicity Oxidase preparation K m (l M ) k cat (s )1 ) a K m (l M ) k cat (s )1 ) K m (l M ) k cat (s )1 ) K m (l M ) k cat (s )1 ) K m (l M ) k cat (s )1 ) transition at I(m M ) b Four-subunit oxidase ND ND 0.6 434 0.9 555 1.4 669 5.9 1031 36 Four-subunit oxidase ND ND 0.3 63 1.6 154 3.6 338 10.5 400 26 purified with F v Four-subunit oxidase ND ND 0.5 88 1.1 270 4.1 384 ND ND 26 + specific F v added Four-subunit oxidase ND ND ND ND ND ND 1.6 555 ND ND 36 + control F v added Two-subunit oxidase ND ND 0.8 254 0.85 263 2.9 288 15.1 336 26 Two-subunit oxidase 1.6 220 3.8 243 ND ND ND ND ND ND 7.4 + specific F v added Four-subunit TM1 c ND ND 0.56 8 1.6 20 7.9 25 ND ND 14.8 Two-subunit TM1 c ND ND 7 30 ND ND ND ND ND ND 14.8 Four-subunit oxidase ND ND 2.8 1000 28.5 474 52.5 100 ND ND 26 vs. c 552 –f d a The K m value is taken from the high-affinity phase and k cat from the low-affinity phase, whenever kinetics are biphasic. b On lowering the ionic strength, transition from monophasic to biphasic kinetics is observed at specified ionic strength (I). c Triple mutant TM1 (E126Q, D135N, D178N) in subunit II. d Data taken from V. Drosou & B. Ludwig, unpublished results. Fig. 3. Eadie–Hofstee plots (A and B) representing horse heart cyto- chrome c oxidation by the two-subunit oxidase at different ionic strength conditions. Steady-state kinetics were determined spectrophotometri- cally at 25 °C. 2984 V. Drosou et al.(Eur. J. Biochem. 269) Ó FEBS 2002 monoclonal IgG directed against a subunit II epitope [22], may be added in a 3 : 1 molar excess to purified oxidase both as a four- or a two-subunit complex. Alternatively, F v may be used to affinity-purify the four-subunit oxidase from solubilized membranes, yielding a stable 1 : 1 complex which was instrumental in the structure determination of the P. denitrificans oxidase [11]. Table 3 indicates that in all cases the F v fragment, present with or added to the enzyme, induced a decrease in the transition point to biphasic kinetics. To some extent, individual effects appear to be additive when following this shift from the four-subunit to the two-subunit enzyme, and to the F v -complexed oxidase lacking the two smallest subunits. In a control reaction employing an unspecific F v protein not recognizing any oxidase epitope [26], wild-type beha- viour ensued. It should also been noted that under true biphasic conditions (26 m M ), kinetic parameters for the F v -complexed oxidase point at a somewhat diminished overall catalytic efficiency of this enzyme form (see Table 3), although, with the exception of the F v -complexed two- subunit oxidase, the high affinity K m values are comparable. While all the above mentioned experiments were per- formed with the commercially available horse heart cyto- chrome c, the heterologous expression of a soluble c-type cytochrome fragment, c 552 -f, will allow to probe this bacterial oxidase with its homologous electron donor derived from P. denitrificans [27–30]. With regard to reaction kinetics with the four-subunit oxidase complex, this soluble bacterial cytochrome fragment is a competent donor to oxidase (V. Drosou & B. Ludwig, unpublished results), and more importantly it is characterized by biphasic Eadie–Hofstee plots once the ionic strength drops to 26 m M or below (see Table 3, last row). DISCUSSION Extent of the acidic patch on subunit II involved in the cytochrome c docking reaction A two-step model has been proposed to describe the docking of the membrane-embedded oxidase with its soluble sub- strate cytochrome c. In a first step governed by long–range electrostatic interaction mediated by oppositely charged surfaces on either protein, a preorientation of both redox partners is obtained, which is followed by a fine-tuning mediated by hydrophobic surfaces to aquire a docking conformation for optimal electron transfer [14,32]. A strong, positive surface potential for the mitochondrial electron donor, cytochrome c, is evident, while several acidic residues have been suggested to participate in docking on a negatively charged patch located mostly on subunit II above the first electron acceptor in oxidase, the Cu A center (see introduc- tion). A bell-shaped dependency of the turnover number on ionic strength of the assay medium (see also Fig. 2) has been taken as initial experimental evidence that protein surface charges get progressively shielded by increasing the ionic strength of the medium. Under turnover conditions, an optimal salt concentration results from a compromise of the association and the dissociation rates for cytochrome c. From a previous mutagenesis study [13], a partial contribution of a few acidic residues on subunits I and III to the acidic docking site on the periplasmic face of the P. denitrificans oxidase appeared likely. Making use of the fact that this bacterial enzyme can be isolated both as a four- and a two-subunit complex without major kinetic defects (see Fig. 2, and below), a distinct decrease (by 20 m M ) in the ionic strength maximum for the two- subunit wild-type complex confirms the contribution of additional charge(s) located on the two further subunits of the native oxidase. In focussing on the main interaction domain on subunit II, we introduced additional mutations in exposed residues in the relevant area above the Cu A site (see Fig. 1 and Table 2), to estimate the extent of the acidic region responsible for cytochrome c docking. While no direct structural information is at hand for the docked complex, the interaction domain for cytochrome c on the cyto- chrome bc 1 complex of yeast turned out to be confined to a few residues only [33]. Both the mutants H119N and N160D (Table 2) show wild-type characteristics, along with an ionic strength optimum at 56 m M . For H119N this is not surprising since no charge change results. In position N160 an additional negative charge was introduced, but available kinetic parameters suggest that this mutant, despite its higher negative surface potential, does not provide a more potent docking site for its substrate. This observation may be explained by the fact that this residue is located too far out from the actual electron entry site W121 (see below). Mutants E140Q, E142Q, D146N, and P196G all show a shift in the ionic strength optimum to 46 m M , providing first evidence that these residues are involved in substrate binding. They were characterized at 56 m M and at 36 m M ; in the latter condition, clear biphasic kinetics were recorded (see below). Mutants D135N and D178N and the triple mutant TM1 have already been characterized as bona fide docking mutants in the past [13] but were re-analyzed side by side with the other mutants generated here. Three of these positions were changed from an acidic side chain into the corresponding amide derivative, yielding unequivocal evi- dence for their contribution in the cytochrome c oxidation reaction. Compared to wild-type, they show some changes in K m ,butatthesametimealsoink cat . When biphasic reactions are obtained at 36 m M ionic strength, the tendency increases for a more pronounced rise in the K m value, but a concomitant loss in k cat cannot be overlooked under this condition either. This decrease of turnover numbers may partly be explained by the fact that for the purpose of a uniform comparison, these mutants were measured at 36 m M and 56 m M whereas the individual optimal ionic strength was found to be at 46 m M in some cases. When presteady-state kinetics are measured at 140 m M ionic strength for this set of mutants, it is evident that a parallel trend, even though not always in a quantitative manner, is seen for E142, D146N, and E140Q (Table 2). The double mutant H119I/Q120I should lead to an increase of the hydrophobic free energy, and its K m value is increased (along with a decrease of k cat ), which means that substrate binding is influenced. Since the single mutant H119N showed wild-type behaviour, the position Q120 is most likely responsible for the observed effects. The interpretation of the low-affinity K m values (not given) is not straightforward since the explanation for this phase is still hypothetical (see below). However, the same general trend for both the high-affinity and the low-affinity K m values is observed. Ó FEBS 2002 Cytochrome c docking site (Eur. J. Biochem. 269) 2985 Taken together with our earlier data [13], this study now defines an extensive area of exposed acidic residues on subunit II which are involved in the initial docking (see above) of the horse heart cytochrome c. In viewing down the axis from W121 to the Cu A center as in Fig. 1, a lobe of three carboxylate side groups (D146, E140, D159), with a minor contribution from E142, extends to the edge of the presumed interaction site. A more central region, closer to W121, is made up of residues D135, E126, and D178 (as modified together in the triple mutant TM1). Further residues important for interaction in this latter lobe include Q120, and possibly P196. This docking site model includes the four homologous positions of acidic residues considered most effective also in the Rhodobacter spheroides heme aa 3 oxidase [34]. Experiments replacing the mitochondrial cytochrome c with a fragment of the homologous bacterial electron donor, cytochrome c 552 of P. denitrificans [27,28] confirm that all of the above mentioned residues on oxidase are also involved in this docking reaction, while some additional ones appear specific for the bacterial donor protein (for details, see V. Drosou & B. Ludwig, unpublished results). From this we conclude that the surface area on oxidase, covered by the bacterial cytochrome c, is at least as large as that for the mitochondrial protein. Specificity of the electron entry site into oxidase Previous mutagenesis data on the P. denitrificans [14] and on the Rh. spheroides [34] oxidase clearly indicated that the tryptophan at position 121 is of crucial importance for electron transfer from cytochrome c to the Cu A center in oxidase. Being located approx. 5 A ˚ above the metal center, it is followed in sequence by another aromatic side chain, Y122. Table 1 summarizes the kinetic effects of single mutations in either residue, and of several double mutants, indicating that in no case any major changes on K m , resp., on affinity towards the substrate, occur. However, whenever a W121 mutation is introduced, k cat is drastically diminished to a few percent residual activity for aromatic side chain replacements, and even lower for aliphatic ones. On the contrary, exchanges in the neigh- bouring aromatic residue, Y122, only lead to moderate or no activity changes at all. Double mutants like the W121Q/Y122Q do not fall below the single W121Q activity, i.e. its (already low) residual electron transfer activity is not maintained by the neighbouring tyrosine, while the W121F/Y122F mutant activity may be viewed as a commitment of the tyrosine residue to support the (somewhat higher) residual activity of the W121F single mutant. Pre-steady-state kinetics again fully support the turnover data, showing that for some cases a bimolecular rate in the electron transfer reaction is no longer meas- urable (see Table 1). We conclude that a tryptophan is strictly required in this position to accept electrons from cytochrome c, most likely for steric reasons, since virtually no other residue, not even another aromate, neither in this position nor an adjacent position, is apt for maintaining this role. This statement seems to hold true for further alternative positions suggested from computational docking studies (L. Dutton, Johnson Foundation, Philadelphia, PA, USA, personal communi- cation) as potential entry site: mutations in an exposed tyrosine (Y226F; see Table 1) and in a histidine (H119N; Table 2) show wild-type kinetics. Biphasic steady-state kinetics Non-linear kinetics have been observed for cytochrome c oxidation (see introduction) in many experimental systems. Generally speaking, higher ionic strength conditions result in monophasic plots in a typical Eadie–Hofstee presenta- tion, whereas experiments at lower ionic strength may lead to biphasic kinetics. This effect is exemplified in Fig. 3 for the isolated two-subunit oxidase complex in going from I ¼ 56 to I ¼ 15 m M ionic strength assay conditions, where the transition to biphasicity occurs at 26 m M .Basedonthis observation, we further examine the bacterial oxidase and specify a number of widely differing conditions (see Table 3) to manipulate this transition point from mono- to biphasic behaviour. Subunit composition of the oxidase complex, as already discussed above in terms of ionic strength optimum of cytochrome c oxidation, is an experimental criterion for differentiation: the two-subunit complex reaction becomes biphasic at a lower salt concentrations when compared to the four-subunit enzyme (see Table 3). Loss of charged (acidic) side chains, either in many single mutations or in the triple mutant TM1 (Table 3), down- shifts the transition considerably, also in the context of the above subunit criterion. Binding of F v to the subunit II epitope has a profound effect on the transition. As outlined in Table 3, this cannot be explained by the purification method since this effect occurs both for a F v (affinity chromatography protocol) preparation as well as for a conventionally isolated enzyme incubated with a threefold molar excess of F v prior to the kinetic measurement. Moreover, the effect is specific for the particular epitope/antibody, and cannot be mimicked by addition of a F v antibody preparation lacking any oxidase affinity. This kinetic phenomenon is difficult to rationalize since the epitope is located on a site of subunit II, opposite of the presumed docking area for cytochrome c [11], and a direct competition with substrate therefore appears unlikely. We also note that both the K m and the k cat of oxidase are appreciably perturbed under most conditions when the specific F v is present. At least two possible explanations may be given at this point, either a slight conformational ÔfreezingÕ effect due to the tight F v binding, or a general disturbance of the surface potential of the hydrophilic region of this subunit. Different donor molecules do cause such shifts as well. Comparing the standard horse heart cytochrome c with the homologous bacterial donor, c 552 (employed as a soluble fragment; Table 3, last line), the transition point is lowered for the four-subunit complex reacting with the Paracoccus donor. From the above collection of examples (which are largely descriptive in nature), it is evident that so far we cannot find any in vitro conditions under which cytochrome c oxidation proceeds in a strictly monophasic manner, apart from increasing ionic strength. Whenever the ionic strength in the assay medium is adequately reduced, nonhyperbolic Michaelis–Menten kinetics can be obtained. However, in this investigation we have been able to exclude that this general feature depends on (a) the presence of subunits III 2986 V. Drosou et al.(Eur. J. Biochem. 269) Ó FEBS 2002 andIVintheParacoccus enzyme, and by inference on the presence of cytoplasmically coded subunits of the mito- chondrial enzyme as well, and (b) on differences in purification strategies. We also have no evidence that biphasicity is a consequence of a potential second binding site for cytochrome c, as recently again suggested for the mitochondrial enzyme on the basis of crosslinking experi- ments [35] and theoretical considerations [36], which, however, are in contradiction to early evidence, obtained on spectroscopic grounds [37], favouring a single functional binding site: Several attempts to eliminate a hypothetical second site have been made here for the bacterial enzyme by stripping subunits III and IV off the native complex, and by further destroying a large part of the acidic lobe(s) of the docking site of subunit II in the TM1 mutant. Nevertheless, even the latter construct, as a severely crippled two-subunit complex, displays biphasic kinetics. Inspecting all the above data, it appears that the transition point (to biphasic behaviour), as a general trend, lies below the ionic strength value for the turnover maximum. We may speculate that the kinetic phenomenon of biphasicity is simply caused, in mechanistic terms, by steric interference between oxidized cytochrome c (with a sluggish off-rate to dissociate from the enzyme), and the next incoming reduced cytochrome c molecule, both com- peting for the docking site under turnover conditions [15]. In this context it is interesting to note that even for a covalently linked cytochrome c domain, as present, e.g. in the caa 3 oxidase of B. subtilis, biphasic reaction kinetics have been reported in the ascorbate/tetramethyl-p-phenylenediamine assay [38]. Thus, the observed low ionic strength nonhy- perbolic Michaelis–Menten kinetics may not be solely due to changes in the initial ferrocytochrome c concentration, and rather are an intrinsic enzymic property ensuing from the mechanistic details of the cytochrome oxidase reaction. ACKNOWLEDGEMENTS We are grateful to Maurizio Brunori and Oliver Richter for helpful criticism, to Andrea Hermann and Hans-Werner Mu ¨ ller for excellent technical assistance, and thank Petra Hellwig for help with the Cu A redox potential determination. This work was supported by Deutsche Forschungsgemeinschaft (SFB 472) and Fonds der Chemischen Industrie, by Conferenza dei Rettori delle Universita ` Italiane, and Deutscher Akademischer Austauschdienst (DAAD Vigoni Program). REFERENCES 1. Trumpower, B.L. & Gennis, R.B. (1994) Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to trans- membrane proton translocation. Annu.Rev.Biochem.63, 675– 716. 2. deGier,J W.L.,Lu ¨ bben, M., Reijnders, W.N.M., Tipker, C.A., Slotboom, D J., van Spanning, R.J.M., Stouthamer, A.H. & van der Oost, J. (1994) The terminal oxidases of Paracoccus denitrificans. Mol. Microbiol. 13, 183–196. 3. Michel, H., Behr, J., Harrenga, A. & Kannt, A. (1998) Cyto- chrome c oxidase: structure and spectroscopy. Annu.Rev.Biomol. Struct. 27, 329–356. 4. Ludwig, B., Bender, E., Arnold, S., Hu ¨ ttemann,M.,Lee,I.& Kadenbach, B. (2001) Cytochrome c oxidaseandtheregulationof oxidative phosphorylation. Chembiochem. 2, 392–403. 5. Staudenmeyer, N., Ng, S., Smith, M.B. & Millet, F. (1977) Effects of specific trifluoroacatylation of individual cytochrome c lysines on the reaction with cytochrome oxidase. Biochemistry 16, 600– 604. 6. Ferguson-Miller, S., Brautigan, D. & Margoliash, E. (1978) Definition of cytochrome c binding domains by chemical mod- ification. J. Biol. Chem. 253, 149–159. 7. Rieder, R. & Bosshard, H.R. (1980) Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc 1 and cytochrome c 1 . J. Biol. Chem. 255, 4732–4739. 8. Taha, T.S.M. & Ferguson-Miller, S. (1992) Interaction of cyto- chrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent. Biochemistry 31, 9090–9097. 9. Bisson, R., Steffens, G.C.M., Capaldi, R.A. & Buse, G. (1982) Mapping of the cytochrome c binding site on cytochrome c oxi- dase. FEBS Lett. 144, 359–363. 10. Lappalainen, P., Watmough, N.J., Greenwood, C. & Saraste, M. (1995) Electron transfer between cytochrome c and the isolated Cu A domain: Identification of substrate-binding residues in cyto- chrome c oxidase. Biochemistry 34, 5824–5830. 11. Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Struc- ture at 2.8 A ˚ resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376, 660–669. 12. Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R. & Yoshikawa, S. (1996) The whole structure of the 13-subunit oxi- dized cytochrome c oxidase at 2.8 A ˚ . Science 272, 1136–1144. 13. Witt,H.,Malatesta,F.,Nicoletti,F.,Brunori,M.&Ludwig,B. (1998) Cytochrome c binding site on cytochrome c oxidase in Paracoccus denitrificans. Eur. J.Biochem. 251, 367–373. 14. Witt,H.,Malatesta,F.,Nicoletti,F.,Brunori,M.&Ludwig,B. (1998) Tryptophan 121 of subunit II is the electron entry site to cytochrome c oxidase in Paracoccus denitrificans – involvement of a hydrophobic patch in the docking reaction. J. Biol. Chem. 273, 5132–5136. 15. Ferguson-Miller, S., Brautigan, D. & Margoliash, E. (1976) Cor- relation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cyto- chrome c oxidase. J.Biol. Chem. 251, 1104–1115. 16. Speck, S.H., Dye, D. & Margoliash, E. (1984) Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase. Proc. Natl Acad. Sci. USA 81, 347–351. 17. Brzezinski, P. & Malmstro ¨ m, B.G. (1986) Electron-transport-dri- ven proton pumps display nonhyperbolic kinetics: simulation of the steady-state kinetics of cytochrome c oxidase. Proc. Natl Acad. Sci. USA 83, 4282–4286. 18. Witt, H., Zickermann, V. & Ludwig, B. (1995) Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c. Biochim. Biophys. Acta 1230, 74–76. 19. Ludwig, B. (1986) Cytochrome c oxidase from Paracoccus deni- trificans. Methods Enzymol. 126, 153–159. 20. Gerhus, E., Steinru ¨ cke, P. & Ludwig, B. (1990) Paracoccus deni- trificans cytochrome c 1 gene replacement mutants. J. Bacteriol. 172, 2392–2400. 21. Hendler, R.W., Pardhasaradi, K., Reynafarje, B. & Ludwig, B. (1991) Comparison of energy-transducing capabilities of two- and three-subunit cytochromes aa 3 from Paracoccus denitrificans and the 13-subunit beef heart enzyme. Biophys. J. 60, 415–423. 22. Kleymann, G., Ostermeier, C., Ludwig, B., Skerra, A. & Michel, H. (1995) Engineered F v fragments as a tool for the one-step purification of integral multisubunit membrane protein com- plexes. Biotechnology N.Y. 13, 155–160. 23. Pfitzner, U., Odenwald, A., Ostermann, T., Weingard, L., Ludwig, B. & Richter, O M.H. (1998) Cytochrome c oxidase (heme aa 3 ) Ó FEBS 2002 Cytochrome c docking site (Eur. J. Biochem. 269) 2987 from Paracoccus denitrificans: analysis of mutations in putative proton channels of subunit I. J. Bioenerg. Biomembr. 30, 89–97. 24. Ludwig, B. & Schatz, G. (1980) A two-subunit cytochrome c oxidase (cytochrome aa 3 )fromParacoccus denitrificans. Proc. Natl Acad. Sci. USA 77, 196–200. 25. Nicoletti,F.,Witt,H.,Ludwig,B.,Brunori,M.&Malatesta,F. (1998) Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes. Biochim. Biophys. Acta 1365, 393–403. 26. Ostermann, T. (2000) Herstellung von Fv-Antiko ¨ rperfragmenten als Hilfsmittel zur Reinigung und Kokristallisation membranesta ¨ ndiger Chinoloxidasen von Escherichia coli und Paracoccus denitrificans. PhD Thesis, University of Frankfurt, Germany. 27. Turba, A., Jetzek, M. & Ludwig, B. (1995) Purification of the cytochrome c 552 of Paracoccus denitrificans and sequence analysis of the gene. Eur. J. Biochem. 231, 259–265. 28. Reincke, B., Tho ¨ ny-Meyer, L., Dannehl, C., Odenwald, A., Aidim, M., Witt, H., Ru ¨ terjans, H. & Ludwig, B. (1999) Hetero- logous expression of soluble fragments of cytochrome c 552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase. Biochim. Biophys. Acta 411, 114–120. 29. Reincke, B., Pe ´ rez, C., Pristovsek, P., Lu ¨ cke, C., Ludwig, C., Lo ¨ hr, F.,Rogov,V.,Ludwig,B.&Ru ¨ terjans, H. (2001) Solution struc- ture and dynamics of the functional domain of Paracoccus deni- trificans c 552 in both redox states. Biochemistry 40, 12312–12320. 30. Harrenga, A., Reincke, B., Ru ¨ terjans, H., Ludwig, B. & Michel, H. (2000) Structure of the soluble domain of cytochrome c 552 from Paracoccus denitrificans in the oxidized and reduced states. J. Mol. Biol. 295, 667–678. 31. Povray, E. (1994) Schwan Ray Tracing for the Macintosh CD. Waite Group Press, Corte Madera, CA, USA. 32. McLendon, G. & Hake, R. (1992) Interprotein electron transfer. Chem. Rev. 92, 481–490. 33. Lange, C. & Hunte, C. (2002) Crystal structure of the yeast cytochrome bc 1 complex with its bound substrate cytochrome c. Proc. Natl Acad. Sci. USA 99, 2800–2805. 34. Zhen, Y., Hoganson, C.W., Babcock, G.T. & Ferguson-Miller, S. (1999) Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic char- acterization of surface mutants in subunit II of Rhodobacter spheroides cytochrome aa 3 . J. Biol. Chem. 274, 38032–38041. 35. Sampson, V. & Alleyne, T. (2001) Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme turnover. Eur. J. Biochem. 268, 6534–6544. 36. Roberts, V.A. & Pique, M.E. (1999) Definition of the interaction domain for cytochrome c on cytochrome c oxidase. III. Prediction of the docked complex by a complete, systematic search. J. Biol. Chem. 274, 38051–38060. 37. Michel, B. & Bosshard, H.R. (1984) Spectroscopic analysis of the interaction beween cytochrome c and cytochrome c oxidase. J.Biol. Chem. 259, 10085–10091. 38. Assempour, M., Lim, D. & Hill, B.C. (1998) Electron transfer kinetics during the reduction and turnover of the cytochrome caa 3 complex from Bacillus subtilis. Biochemistry 37, 9991–9998. 2988 V. Drosou et al.(Eur. J. Biochem. 269) Ó FEBS 2002 . Mutations in the docking site for cytochrome c on the Paracoccus heme aa 3 oxidase Electron entry and kinetic phases of the reaction Viktoria. for a second cytochrome c binding site on oxidase. Keywords: Paracoccus denitrificans; cytochrome c oxidase; docking site; electron transfer; biphasic kinetics. Cytochrome