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• W2059797734 abstract "Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ∼70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO. Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ∼70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO. Electron transfer reactions between and within proteins bearing redox-active constituents are abundant in the cell. Most physiological electron transfer reactions take place in the sub-millisecond time scale (1Page C.C. Moser C.C. Chen X. Dutton P.L. Nature. 1999; 402: 47-52Crossref PubMed Scopus (1470) Google Scholar). Experimentally, it is not possible to study the dynamics of such reactions by mechanically mixing the reactants (e.g. stopped-flow spectroscopy). Such dynamics may, however, be studied by the use of light pulses. This approach is obvious for systems where light is a natural trigger, such as photosynthetic systems, but in some cases this approach can also be used with other pigmented proteins. The method we intend to develop relies on the fact that the gaseous ligand carbon monoxide (CO) 4The abbreviations used are: CO, carbon monoxide; cyt., cytochrome; cm-cyt. c, carboxymethylated cytochrome c; ferri-cyt. c, ferricytochrome c; Mb, myoglobin. 4The abbreviations used are: CO, carbon monoxide; cyt., cytochrome; cm-cyt. c, carboxymethylated cytochrome c; ferri-cyt. c, ferricytochrome c; Mb, myoglobin. has a high affinity for ferrous heme proteins but does not bind to the ferric heme counterpart. Therefore, the effect of CO is to increase the apparent redox potential of any heme protein that has a vacant sixth coordination position and is thus able to bind this ligand. The CO molecule can be photo-cleaved from heme with a quantum yield close to unity (i.e. one CO molecule photodissociated for each photon absorbed by the heme group). Thus, a light pulse will dissociate the CO from the heme group, leaving this as a 5-coordinate high spin ferrous species. This has the effect of rapidly switching the redox potential of the protein that stabilizes the ferrous form (the CO adduct) into a good electron donor (5-coordinate ferrous heme). Provided CO does not rebind rapidly (i.e. >10–5 s), photo-induced electron transfer can thus take place. These features have been exploited to investigate intraprotein electron transfer from photodissociated 5-coordinated heme a3 in the active site of native cytochrome c oxidase, where CO acts as an inhibitor (2Boelens R. Wever R. Van Gelder B.F. Biochim. Biophys. Acta. 1982; 682: 264-272Crossref PubMed Scopus (63) Google Scholar), and recently used to determine a rate as high as ∼109 s–1 (3Pilet E. Jasaitis A. Liebl U. Vos M.H. Proc. Natl. Acad. Sci. U. S. A. 2004; : 16198-16203Crossref PubMed Scopus (68) Google Scholar). This study focuses on cytochrome c (cyt. c), a ubiquitous, small, soluble electron transfer protein carrying one heme cofactor. In native cyt. c, the heme iron is coordinated by two internal axial ligands, His-18 and Met-80, and cannot bind CO. However in methionine-modified forms (4Brzezinski P. Wilson M.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6176-6179Crossref PubMed Scopus (35) Google Scholar, 5Larsen R.W. Biochim. Biophys. Acta. 2003; 1619: 15-22Crossref PubMed Scopus (4) Google Scholar), like carboxymethylated cytochrome c (cm-cyt. c), chemical modification of Met-80 precludes formation of the heme iron–Met-80 bond and allows CO to bind. In a sizeable fraction of cm-cyt. c (12–26% depending on the modification protocol (6Silkstone G. Jasaitis A. Vos M.H. Wilson M.T. Dalton Trans. 2005; 21: 3489-3494Crossref Scopus (22) Google Scholar)), CO rebinding is slow enough to allow flash-induced oxidation of the heme. This technique has been successfully used to study electron transfer from cm-cyt. c to cytochrome c oxidase (4Brzezinski P. Wilson M.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6176-6179Crossref PubMed Scopus (35) Google Scholar) and to plastocyanin (7Karpefors M. Wilson M.T. Brzezinski P. Biochim. Biophys. Acta. 1998; 1364: 385-389Crossref PubMed Scopus (9) Google Scholar). These studies have provided proof of principle for the method, but the use of cm-cyt. c, although useful, is not ideal. This is primarily because the carboxymethylation reaction is not specific and can also lead to chemical modification of important “docking” residues on the surface of the protein. With the objective of developing a method for studying inter-protein electron transfer that employs proteins in “quasi-native” states, we have made a series of Met-80 mutants of yeast iso-1-cytochrome c, namely M80A, M80S, M80T, M80D, and M80E (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar). Such mutants bind CO (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar, 9Bren K.L. Gray H.B. J. Am. Chem. Soc. 1993; 115: 10382-10383Crossref Scopus (11) Google Scholar). Fig. 1 shows the solution structure of yeast cyano-M80A ferri-cyt. c (10Bren K.L. Gray H.B. Banci L. Bertini I. Turano P. J. Am. Chem. Soc. 1995; 117: 8067-8073Crossref Scopus (57) Google Scholar) with labeled and some important amino acid residues that line the distal heme pocket where CO would normally bind to the ferrous heme iron (see under “Discussion”). The replacement residues for the native Met-80 were selected as those that were not expected to coordinate to the heme iron, especially in the ferrous redox state. However, for electron transfer experiments, these mutants have one drawback, namely that the apparent quantum yield (φ) for photon-induced CO escape from the protein is very small (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar). This is because of rapid and efficient “geminate” recombination of CO and the heme. We have recently shown that in cm-cyt. c such recombination is multiphasic and predominantly takes place on the picosecond time scale (6Silkstone G. Jasaitis A. Vos M.H. Wilson M.T. Dalton Trans. 2005; 21: 3489-3494Crossref Scopus (22) Google Scholar), i.e. much faster than inter-protein electron transfer (typically ∼105 s–1). In contrast to NO recombination, extensive geminate recombination of CO and heme is seldom observed in native natural ligand-binding proteins like myoglobin (Mb), and in particular not on the sub-nanosecond time scale (11Vos M.H. Martin J.-L. Biochim. Biophys. Acta. 1999; 1411: 1-20Crossref PubMed Scopus (106) Google Scholar). The fact that such recombination, by contrast, dominates in cyt. c can be understood in general terms by the absence of a “natural” ligand exchange pathway and by a different regime of protein dynamics in this electron transfer protein. For efficient low driving force electron transfer to occur, the reorganization energy of the environment of the redox partner (in the case of cyt. c the heme) should be small. Indeed, in cyt. c the heme group is surrounded by a hydrophobic and relatively rigid pocket (12Flynn P.F. Bieber Urbauer R.J. Zhang H. Lee A.L. Wand A.J. Biochemistry. 2001; 40: 6559-6569Crossref PubMed Scopus (41) Google Scholar, 13Louie G.V. Brayer G.D. J. Mol. Biol. 1990; 214: 527-555Crossref PubMed Scopus (390) Google Scholar), and the protein contribution to the reorganization energy of cyt. c is thought to be weak (14Muegge I. Qi P.X. Wand A.J. Chu Z.T. Warshel A. J. Phys. Chem. 1997; 101: 825-836Crossref Scopus (205) Google Scholar, 15Simonson T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6544-6549Crossref PubMed Scopus (86) Google Scholar). Structural flexibility is not advantageous for that function. In contrast, proteins where ligand transfer is essential, structural flexibility is required to create temporary passages through which ligands may diffuse. In a general sense, the constructed CO-binding cyt. c mutants offer a unique chance to study these dynamics-function relations by comparison with functional ligand-binding proteins like Mb. Studying the picosecond/nanosecond recombination dynamics of CO and heme in cyt. c proteins incorporating an M80X mutation can give insight into the heme environment. In a similar way, in wild type and mutant Mb, multiphasic picosecond heme-NO recombination kinetics have proven a sensitive tool for assessing the dynamic, steric, and electrostatic role of the heme environment (16Martin J.-L. Vos M.H. Methods Enzymol. 1994; 232: 416-430Crossref PubMed Scopus (39) Google Scholar, 17Petrich J.W. Lambry J.-C. Balasubramanian S. Lambright D.G. Boxer S.G. Martin J.-L. J. Mol. Biol. 1994; 238: 437-444Crossref PubMed Scopus (54) Google Scholar, 18Ikeda-Saito M. Dou Y. Yonetani T. Olson J.S. Li T. Regan R. Gibson Q.H. J. Biol. Chem. 1993; 268: 6855-6857Abstract Full Text PDF PubMed Google Scholar, 19Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar, 20Olson J.S. Phillips Jr., G.N. J. Biol. Chem. 1996; 271: 17593-17596Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 21Carlson M.L. Regan R. Elber R. Li H. Phillips Jr., G.N. Olson J. S Gibson Q.H. Biochemistry. 1994; 33: 10597-10606Crossref PubMed Scopus (70) Google Scholar, 22Kim S. Lim M. J. Am. Chem. Soc. 2005; 127: 8908-8909Crossref PubMed Scopus (30) Google Scholar). As mentioned above, in Mb, CO geminate recombination is usually less extensive and takes place on a much longer time scale (23Sugimoto T. Unno M. Shiro Y. Dou Y. Ikeda-Saito M. Biophys. J. 1998; 75: 2188-2194Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 24Nienhaus K. Deng P. Olson J.S. Warren J.J. Nienhaus G.U. J. Biol. Chem. 2003; 278: 42532-42544Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Cao W. Ye X. Georgiev G.Y. Berezhna S. Sjodin T. Demidov A.A. Wang W. Sage J.T. Champion P.M. Biochemistry. 2004; 43: 7017-7027Crossref PubMed Scopus (31) Google Scholar) and in the native protein in a monophasic way (τ =∼150 ns). To gain insight into the dynamic properties of the hydrophobic heme-binding core of cyt. c and in particular to investigate ways to optimize the escape probability of photodissociated CO by modifying the heme environment, we have measured CO recombination kinetics in a number of cyt. c mutants. Using spectrally resolved femtosecond spectroscopy, we also determined the heme spectra associated with different decay phases, which are indicators of the interaction of the heme with its environment, and in particular with the dissociated nearby ligand. This initial study concerns a limited number of mutants. By substituting Met-80 the idea was to investigate the effects of introducing both steric and hydrophobic constraints. In addition to proteins bearing Ala, Ser, and Asp at this position, which were previously shown to have a low millisecond CO escape efficiency (φ < 0.05) (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar), we investigate the effect of the more bulky, charged residue Arg. Furthermore, starting from the M80D mutation, the effect of the additional substitution of the close-lying Phe-82 by a smaller residue (Asp or Gly) is explored. Mutagenesis—Site-directed mutagenesis, protein expression in Escherichia coli, and purification of yeast iso-1-cytochrome c mutants bearing single (M80A, M80S, M80D, or M80R) mutations in the heme environment were performed as described previously (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar). The double mutations (M80D/F82D or M80D/F82G) were introduced using a single-step PCR approach (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar), and in addition using the M80D mutated pBPCYC1(wt)/3 plasmid construct as template DNA. In these proteins, the Cys-102 residue is also changed to a Thr to prevent dimerization through formation of disulfide bridges. Sample Preparation for Ultrafast Spectroscopy—The samples were prepared to a heme concentration of ∼50 μm in a gas-tight optical cell with an optical path length of 1 mm. Unless indicated otherwise, the buffer was 20 mm Hepes, pH 8.0. For the deoxy form, the samples were de-gassed and reduced with 1 mm sodium dithionite. For the carboxy form, the deoxy samples were equilibrated with 1 atm (1 atm = 101.3 kPa) CO. For the nitrosyl form, de-gassed samples were reduced with 10 mm sodium ascorbate and equilibrated with 0.01 atm NO. For the oxy form, 10 mm sodium ascorbate was added to an air-equilibrated sample. Ultrafast Spectroscopy—Multicolor femtosecond absorption spectroscopy (16Martin J.-L. Vos M.H. Methods Enzymol. 1994; 232: 416-430Crossref PubMed Scopus (39) Google Scholar) was performed with a 30-fs pump pulse centered at 565 nm and a <30-fs white light continuum probe pulse, at a repetition rate of 30 Hz. Full spectra of the test and reference beams were recorded using a combination of a polychromator and a CCD camera. All experiments were carried out at 21 °C. The sample was continuously moved perpendicular to the beams to ensure sample renewal between shots. Basic data matrix manipulations and presentation were performed using Matlab (The Mathworks, South Natick, MA). The absorbance changes were treated using the SPLYMOD algorithm (26Provencher S.W. Vogel R.H. Deuflhard P. Hairer E. Numerical Treatment of Inverse Problems in Differential and Integral Equations. 2. Birkhauser, Boston1983: 304-319Google Scholar), with a Matlab interface (27Morgan J.E. Verkhovsky M.I. Puustinen A. Wikström M. Biochemistry. 1995; 34: 15633-15637Crossref PubMed Scopus (57) Google Scholar). Determination of Quantum Yield of CO Escape—The apparent quantum yield φ of dissociated CO on the millisecond time scale was also measured by the “pulsed” method developed by Brunori and co-workers (28Brunori M. Giacometti G.M. Antonini E. Wyman J. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3141-3144Crossref PubMed Scopus (40) Google Scholar) and as described previously (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar). The method determines the value of φ for a given protein relative to a standard, generally Mb, for which the value is assigned as φ = 1(i.e. unity quantum yield). Bimolecular Binding of Gaseous Ligands to the Ferrous Forms of M80A (CO, O2, and NO) and M80D (CO and NO) by Flow Mixing—Protein concentrations were typically 3–5 μm final after flow mixing. The mutant proteins were reduced prior to mixing by additions of sodium dithionite. For the CO and NO experiments, an excess of sodium dithionite was added to remove any dissolved O2 from solution assumed to be present at a concentration of ∼280 μm at 20 °C (29Weiss R.F. Deep-Sea Res. Ocean. Abstr. 1970; 17: 721-735Crossref Scopus (1530) Google Scholar), therefore eliminating competition between gaseous ligands for the ferrous iron (see below for O2). The temperature was maintained at 20 °C in all flow experiments. The observed rates (kobs, s–1) of ligand binding were fitted to single exponential fits and plotted, for each ligand, as a function of their concentration (μm). The data points for each ligand for M80A were then fitted with a rectangular hyperbola (see “Discussion”). The data points for each ligand for M80D were fitted to straight lines (see “Discussion”). Preparation and Calibration of CO Solutions for Bimolecular Binding Studies—Sodium phosphate buffer (100 mm, pH 7) was placed into a glass tonometer, and the solution was degassed thoroughly with vigorous shaking. CO was passed into the tonometer with mild shaking until the gas had completely equilibrated with the solution, and a slightly positive pressure was obtained. The CO solution was transferred from the tonometer to an Agla glass syringe connected to a micrometer screw gauge. A Mb sample of an absolute known concentration (∼10 μm) was prepared using an extinction coefficient of 121,000 m–1 cm–1 at 435 nm for the deoxy form (30Antonini E. Physiol. Rev. 1965; 45: 123-170Crossref PubMed Scopus (135) Google Scholar). The Mb was reduced with a slight excess of sodium dithionite and placed in an ∼3-ml cuvette that had been previously weighed in addition to a suba-seal. The sample was sealed, and no gas phase was present. The cuvette and sample were then re-weighed assuming 1 g = 1 ml. Spectra (375–650 nm) were recorded of the Mb sample titrated with 2-μl additions of the CO solution until saturation of the protein with CO had been achieved (i.e. no further changes in the absorbance on additions of CO). The suba-seal was then removed, and ∼5 ml of pure CO gas was bubbled through the sample so that complete saturation of the solution was achieved. The final spectrum was then recorded. Each addition of 2 μl of CO caused an increase in absorbance at 424 nm, and so the absorbance change at 424 nm minus at 650 nm was plotted against the volume of CO added. The first point on this plot where subsequent additions of CO produced no further change in absorbance was taken as the 1:1 binding of heme with CO (assuming no free heme present), and so with the concentration of Mb exactly known as well as the volume of the solution, the concentration of CO in the sample was deduced and hence that of the stock CO solution. For the stopped-flow reactions of M80A and CO, the stock CO solution was prepared in sodium phosphate buffer (100 mm, pH 7). For the stopped-flow reactions of M80D and CO, the stock CO solution was prepared in sodium acetate buffer (100 mm, pH 4.8), the CO concentration being determined by titration with Mb at pH 7. Preparation of NO Solutions for Bimolecular Binding Studies—The NO solutions for direct use in the stopped-flow apparatus were prepared using stock solutions of Proli NONOate (Axxora) stored in 25 mm NaOH. The concentration of a stock Proli NONOate solution was determined using the extinction coefficient of 8,600 m–1 cm–1 at 248 nm (commonly 12–14 mm). For each Proli NONOate molecule, 2× NO was released on lowering of the pH. A known amount of stock Proli NONOate solution was added to a glass syringe containing either sodium phosphate buffer (100 mm, pH 7) for M80A or sodium acetate buffer (100 mm pH 4.8) for M80D prior to mixing. The highest concentration of NO in solution prior to mixing was ∼1 mm and was therefore 0.5 mm in NO after mixing with protein. Both buffer solutions were de-gassed prior to addition of the stock Proli NONOate solution so as to prevent unwanted side reactions of NO and O2. NO solutions were subsequently diluted with de-gassed buffer solutions. Preparation of Fe2+-M80A and O2 Solutions for Bimolecular Binding Studies—The concentration of dissolved oxygen in sodium phosphate buffer (100 mm, pH 7) was assumed to be ∼280 μm at 20 °C prior to dilution into de-gassed phosphate buffer and flow mixing. M80A was reduced with a minimum amount of sodium dithionite (10–20 μm total) so as to prevent the removal of the O2 being introduced on flow mixing. M80A is very resistant to autooxidation by air, and so can readily form a stable ferrous iron/oxygen adduct. The rate of autooxidation of M80A has been determined as k =∼1.8 days–1 (31Silkstone G. Biophysical Studies on Methionine-80 Mutants of Yeast Iso-1 Cytochrome C, Ph.D. thesis. University of Essex, Colchester, United Kingdom2000Google Scholar). M80S can also form a stable ferrous iron/oxygen adduct with k =∼0.7 days–1. However, all other mutant proteins in this study autooxidize sufficiently quickly so that the oxygen adducts cannot be observed in the time frame of the stopped-flow apparatus. CO Rebinding in Met-80 Single Mutants—Fig. 2A shows the transient spectra in the Soret band region at selected delay times after CO dissociation from the M80A mutant. The general shape of the spectra is typical of a 5-coordinate minus 6-coordinate ferrous heme spectrum (32Wood P.M. Biochim. Biophys. Acta. 1984; 768: 293-317Crossref PubMed Scopus (95) Google Scholar) and denotes a red-shift of the Soret band after photolysis. The initial spectrum decays in a multiphasic time course on the picosecond and early nanosecond time scale, reflecting recombination of CO with the heme. Along with this decay the shape of the spectra also changes, and in particular the induced absorption maximum shifts from 424 nm at t = 1psto ∼432 nm on the hundreds of picoseconds time scale. These features are qualitatively similar to those observed for cm-cyt. c (6Silkstone G. Jasaitis A. Vos M.H. Wilson M.T. Dalton Trans. 2005; 21: 3489-3494Crossref Scopus (22) Google Scholar), and presumably, they reflect relaxation of the heme configuration during the recombination process. The analysis, in terms of spectra associated with exponential decay processes (decay-associated spectra), is shown in Fig. 2B where these relaxation processes are highlighted. In particular, the fastest decay phase, 14 ps, is associated with a much smaller red-shift than the later decay phases, 85 ps and 1.2 ns. In contrast to cm-cyt. c, at t = 4 ns, virtually all CO has recombined (Figs. 2A and 3). This finding is consistent with the reported very low (φ = 0.004) quantum yield of CO escape from the protein (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar) (Table 1) and implies that the geminate heme-CO recombination, which is at the origin of this loss in quantum yield, is completely covered by the 4-ns time window of this study.FIGURE 3CO recombination kinetics in single mutants at position 80 (M80A, ▪; M80D, ◂; M80R, •; M82S, ♦) as measured at 414 nm, near the maximum of the bleaching. The initial amplitudes following photodissociation of CO were normalized to 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Fitted components of CO rebindingMutantPhase 1Phase 2Phase 3A0φτ1A1τ2A2τ3A3pspspsM80A140.20850.7512000.050.000.004aData are from Ref. 8.M80S300.50900.500.000.006aData are from Ref. 8.M80D440.552500.3525000.060.040.04aData are from Ref. 8.M80D/F82D600.402000.3530000.140.110.11M80D/F82G600.402000.3530000.140.110.13M80R800.507000.250.250.31a Data are from Ref. 8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar. Open table in a new tab The transient spectra of the M80S and M80D mutants are qualitatively similar to the M80A mutant (not shown), but the kinetics are somewhat different (Fig. 3). For M80S, the overall picosecond rebinding kinetics are somewhat faster, and in particular no phase having a τ value longer than 100 ps is discerned. For this mutant also virtually all CO rebinds, in agreement with the low φ (Table 1). The kinetics for CO rebinding in M80D need to be described by three distinct exponential phases, as in M80A, but the time constants for all phases are significantly longer, and at t = 4 ns, ∼4% of the CO has not rebound, in good agreement with the reported φ = 0.04 for this mutant (8Silkstone G. Stanway G. Brzezinski P. Wilson M.T. Biophys. Chem. 2002; 98: 65-77Crossref PubMed Scopus (35) Google Scholar) (Table 1). For the M80R mutant, both the transient spectra (Fig. 2C) and the decay kinetics (Fig. 3) stand apart from the Ala, Asp, and Ser mutants. The overall kinetics are relatively slow and can be described by two phases, the fastest of which has a time constant of 80 ps and no sub-50-ps phase. Importantly, the amplitude of the constant, non-rebinding phase (A0), is also much higher, 0.25, which is more than twice that observed in our previous experiments on cm-cyt. c (6Silkstone G. Jasaitis A. Vos M.H. Wilson M.T. Dalton Trans. 2005; 21: 3489-3494Crossref Scopus (22) Google Scholar). In addition, the spectral evolution during the decay is far less pronounced (a moderate effect can be discerned from the decay-associated spectra of the 80-ps component; see Fig. 2D), and the shift feature is much more symmetric than in the other mutants, where the induced absorption band is relatively broad at all delay times. Although for these proteins a steady-state 5-coordinate spectrum cannot be generated for direct comparison (see below), this latter finding suggests that in the M80R the heme rapidly adopts a more relaxed deoxy-like configuration after CO dissociation. Indeed, the spectrum is qualitatively similar to the steady-state CO dissociation spectra for Mb (33Petrich J.W. Poyart C. Martin J.-L. Biochemistry. 1988; 27: 4049-4060Crossref PubMed Scopus (331) Google Scholar) and the c-type heme-undecapeptide microperoxidase-11 (5Larsen R.W. Biochim. Biophys. Acta. 2003; 1619: 15-22Crossref PubMed Scopus (4) Google Scholar). CO Rebinding in Double Mutants—To explore whether one may influence the probability of photodissociated CO escaping into bulk by further modifying the protein environment of the heme, we have generated double mutants in which in addition to the Met-80 to Asp mutation Phe-82 was replaced by the less bulky residues Gly or Asp. Both double mutants displayed very similar kinetics (Table 1), which were substantially slower than for the M80D single mutant (Fig. 4), and also the constant (A0) and non-rebinding phase was significantly higher. These findings indicate that the CO escape pathway indeed involves the close surroundings of Phe-82. Interestingly, the transient spectra are more symmetric for the double mutants (Fig. 4, inset), already within picoseconds after CO dissociation. This indicates that the free volume created by the second mutation allows reduction of the interaction of the dissociated CO with heme. Taken together with the spectral and kinetic properties of M80R mutant (for both the differences with respect to the M80D single mutant are more pronounced), these data strongly suggest a correlation between the transient spectra and the speed and yield of heme-CO rebinding. NO Rebinding—In many natural ligand-binding heme proteins, NO rebinds predominantly on the picosecond time scale and in a multiphasic way that is very sensitive to the structure of the heme pocket (11Vos M.H. Martin J.-L. Biochim. Biophys. Acta. 1999; 1411: 1-20Crossref PubMed Scopus (106) Google Scholar). We have measured the spectral evolution upon excitation of the M80A-NO complex (Fig. 5), and we found that NO rebinding occurs fully in a single exponential phase with a time constant of 7 ps, i.e. in a very similar way as NO rebinding to wild type horse heart cyt. c (34Cianetti S. Kruglik S.G. Vos M.H. Turpin P.-Y. Martin J.-L. Négrerie M. Biochim. Biophys. Acta. 2004; 1658: 218Google Scholar) (where NO can replac" @default.
• W2059797734 created "2016-06-24" @default.
• W2059797734 creator A5036232057 @default.
• W2059797734 creator A5042872815 @default.
• W2059797734 creator A5063917515 @default.
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• W2059797734 date "2007-01-01" @default.
• W2059797734 modified "2023-10-14" @default.
• W2059797734 title "Ligand Dynamics in an Electron Transfer Protein" @default.
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