pH Dependence of Ferricytochrome c ... - ACS Publications

Apr 18, 2017 - during Binding to Cardiolipin Membranes: Evidence for Histidine as ... binding to cardiolipin-containing small unilamellar vesicles wer...
1 downloads 0 Views 998KB Size
Letter pubs.acs.org/JPCL

pH Dependence of Ferricytochrome c Conformational Transitions during Binding to Cardiolipin Membranes: Evidence for Histidine as the Distal Ligand at Neutral pH Bridget Milorey, Dmitry Malyshka, and Reinhard Schweitzer-Stenner* Department of Chemistry, Drexel University, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: The conformational changes of ferricytochrome c upon binding to cardiolipin-containing small unilamellar vesicles were studied at slightly acidic pH using fluorescence, visible circular dichroism, UV−visible absorption, and resonance Raman spectroscopy. The obtained spectroscopic response data suggest a mode of interaction, which is clearly distinct from the binding process observed at neutral pH. Evidence of a reversible and electrostatic binding mechanism under these conditions is provided through binding inhibition in the presence of 150 mM NaCl. Moreover, UV−visible absorption and resonance Raman spectra reveal that the conformational ensemble of membrane bound cytochrome c is dominated by a mixture of conformers with pentacoordinated and hexacoordinated high-spin heme irons, which contrast with the dominance of low-spin species at neutral pH. While our results confirm the L-site binding proposed by Kawai et al., they point to the protonation of a histidine ligand (H33) as conformational trigger.

T

he electron carrier protein cytochrome c has been utilized frequently as a model system for studying electron transfer and protein folding dynamics.1 More recently, it has also been determined to play a pivotal role in the initial steps of a biochemical cascade leading up to mitochondrial apoptosis.2 For performing the latter function the protein undergoes extensive conformational changes, which lead to a gain of peroxidase activity and eventually to the release of the protein from the mitochondrial membrane into the cytosol, where it triggers the Apaf cascade.3,4 The anionic phospholipid cardiolipin (CL) located in the inner mitochondrial membrane is vital for triggering these conformational transitions in that the protein gains the pro-apoptotic function only after binding to CL.4 In light of the two very different and mutually exclusive functions performed by cytochrome c in the intermembrane space of mitochondria, the characterization of this binding process and the extent to which the native and partially unfolded conformations coexist are of paramount biological significance. Multiple groups have performed a plethora of binding studies that revealed that in the presence of anionic lipids such as CL or phosphatidylglycerol (PG), ferricytochrome c undergoes conformational changes upon binding,3−12 but a relationship between proposed modes of cytochrome c binding and adopted conformations has not yet been established. Cytochrome c exhibits a positive charge under physiological conditions, facilitating electrostatic binding to anionic membranes. This electrostatic interaction has been shown to be the driving force for the A-site binding reported by Kinnunen and coworkers.13 Experiments with mutants suggest the involvement of K72, K78, and K86.14 In addition, these researchers reported © XXXX American Chemical Society

evidence of a nonelectrostatic binding process termed C-site binding driven by hydrogen bonding between protonated phosphate groups and the N52 residue of the protein.15 However, this binding process becomes activated only below pH 5, which is outside the range of most cytochrome c binding studies.15 More recently, Kawai et al. reported another electrostatic binding site (termed L),16 which becomes activated only at slightly acidic pH below 7 and was shown to involve residues K22, K25, K27, H33, and H26. Apparently, the corresponding binding process does not compete with Csite binding, but it fully lies in the pH-region where A-site binding has been thought to be operative.13,16 Moreover, several studies have provided evidence of either a protein insertion into the membrane15 or the insertion of one or even two CL tails into the hydrophobic binding pocket of the protein.7,17−21 While it is well established that cytochrome c binding to CLcontaining surfaces involves conformational changes, a thorough characterization of these changes has only recently been pursued. Pletneva and coworkers, by utilizing fluorescence resonance energy transfer (FRET) between fluorescence labeled residues and the heme group, obtained an equilibrium between different ensembles of compact (C) and extended (E) conformations.5,6,22 On the basis of spectroscopic response data, Pandiscia and Schweitzer-Stenner have similarly described the ensemble of CL-bound proteins as a CL-dependent equilibrium between a native-like nonfluorescing (nf) state Received: March 10, 2017 Accepted: April 18, 2017 Published: April 18, 2017 1993

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998

Letter

The Journal of Physical Chemistry Letters

Figure 1. Spectral response data at pH 6.5, 6.7, and 7.4. (a) Total integrated intensities of the fluorescence F-band, (b) difference in positive and negative maxima of the Soret band CD, (c) total integrated intensities of the CT1 band, and (d) total integrated intensities of the CT2 band. All data are plotted as a function of the cardiolipin concentration in μM units. It should be noted that the CL concentration was increased 10-fold for charge transfer measurements (frames c and d) along with the protein concentration to keep the lipid to protein ratios consistent.

and a partially unfolded fluorescing (f) state.23,24 In both conformations the heme iron is in a low-spin state. The addition of NaCl was found to shift this equilibrium toward the nf state rather than inhibiting the initial binding step. This observation suggests that the actual binding step is not predominantly governed by electrostatic interactions. In the f state, the native M80 is replaced by another ligand, which might be either a lysine25 or a histidine side chain.9 However, in the pH range where the C- and L-site binding are proposed to be dominant, the protonation of the non-native histidine ligand (H33) should cause its dissociation from the heme iron and thus the population of a hexa- or pentacoordinated high-spin state. 26−28 We decided to explore this possibility by spectroscopically analyzing the binding of ferricytochrome c to CL-containing membranes at slightly acidic pH. Determining the relationship between binding and structure as a function of pH is of great biological significance in view of the likely interdependence of pH, cytochrome c binding, and modulation of the mitochondrial transmembrane potential.29 Our data provide evidence for a pH induced switch between protein binding sites, followed by a conformational change triggered by histidine protonation. The thus facilitated binding process is electrostatic in nature. Hence, our findings complement recent studies revealing L-site binding16 with information about concomitant changes of the protein’s spin and ligation state, which are pivotal for its function. To probe the binding and associated structural changes of oxidized cytochrome c to small unilamellar vesicles (SUVs) with 20% TOCL/80% DOPC (1,1′,1,2′-tetraoleyolcardiolipin/ 1,2-deoleoyl-sn-glycero-3-phosphocholine) in the proposed regime of L-site binding, we measured the W59 fluorescence, visible Soret band circular dichroism, and optical absorption as a function of cardiolipin concentration at pH 6.5 and 6.7, which are close to the saturation levels and midpoints of the titrations

performed by Kawai et al.16 The size distribution of the SUVs was determined using dynamic light scattering (Figure S1). The observed scattering profiles show that the produced liposomes can be classified as SUVs in the absence of cytochrome c. In accordance with recent observations,16 vesicle fusion at very low CL to protein ratios leads to the formation of gigantic unilamellar vesicles (GUVs) and a concomitant increase in the sample’s turbidity.14 Upon increasing the CL/protein ratio, the liposome size moves back into the SUV regime but remains somewhat larger than in the absence of proteins (Figure S1). These results are in line with what Oellerich et al. observed for the binding of cytochrome c to PG vesicles.9 The measured fluorescence spectra are shown in Figure S2. The W59 fluorescence is diagnostic of tertiary structure changes due to cytochrome c unfolding.30 The very intense band at a peak position between 330 and 350 nm is labeled as the F-band and is assignable to W59 fluorescence. The less intense band at a peak position between 420 to 460 nm is labeled as the P-band. It may be assignable to phosphorescence, although its origin remains to be established. In line with previous practices, we decomposed the F-band into three sub-bands.23 The integrated fluorescence intensity of the F2-band is plotted as a function of the cardiolipin concentration in Figure 1a for both pH values investigated. Corresponding data previously obtained at pH 7.4 are shown for comparison.23 An increase in F-band intensity with increasing cardiolipin concentration is observed at all pH values, which reflects the partial unfolding of the protein and thus the binding of cyt c molecules to liposome surfaces. Surprisingly, the data suggest reduced fluorescence or binding affinity at pH 6.7 (compared with 7.4). At pH 6.5, the fluorescence regains some of the original pH 7.4 intensity. Together, these data imply that the liposome bound protein 1994

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998

Letter

The Journal of Physical Chemistry Letters

reported cytochrome c binding at acidic pH is driven by electrostatics, we measured the W59 fluorescence and the Soret band CD as a function of CL concentration at pH 6.5 in the presence of 150 mM NaCl. As shown in Figures S6, the addition of salt eliminates all spectral changes. This demonstrates that the observed binding is entirely electrostatic and distinct from the binding process at neutral pH24 and also from the C-site binding.15 To gain further insights into structural changes caused by cytochrome c binding to CL liposomes at acidic pH, we measured resonance Raman (RR) spectra of different cytochrome−liposome mixtures. RR spectra of heme proteins provide detailed information about spin, oxidation, and ligation states of heme proteins indicated by specific marker bands.38 In the peculiar case of ferricytochrome−liposome mixtures, nativelike and non-native-like cytochrome c can be distinguished because only the former is subject to photoreduction in the presence of a sufficient amount of potassium ferrocyanide.39 RR spectra of ferricytochrome c at different CL concentrations were measured at pH 6.5. The difference spectra for these data set are shown in Figure S7. A rapid loss of the low-spin component of the ν3 intensity with increasing CL concentrations and a concomitant gain in intensity of specific high-spin components of multiple spin marker bands (ν2, ν3, and ν10) was observed, as revealed by the decomposition of the RR spectrum measured in the presence of 1500 μM CL (Figure 2).

adopts at least three different states in the investigated pH range. Soret band circular dichroism and charge transfer bands in the region between 600 and 750 nm are all excellent indicators of the ligation state of the heme group and the latter’s interaction with the protein environment.31,32 Figures S3 and S4 show the CD spectra and the coinciding UV−visible absorption spectra of ferricytochrome c measured at different CL concentrations. Qualitatively, the observed changes of the CD spectra are similar at the three pH values in that they depict a transition from a couplet (native state and native-like states)33 to a positive Cotton band. Such a change is diagnostic of a replacement of the native M80 ligand and an opening of the heme crevice.34,35 However, the amplitudes of the observed changes as well as the positions of the positive Cotton band are clearly different. A quantitative assessment of the spectral changes can be achieved by plotting the difference ΔΔε between the dichroism at the positive and negative maxima of the couplet as a function of the cardiolipin concentration (Figure 1b). Apparently, the ΔΔε-response data observed at pH 6.5 and 6.7 both clearly differ from corresponding values observed at pH 7.4.23 The respective values for pH 6.7 are slightly less pronounced than those observed at 6.5, which, in line with the trend indicated by the above pH dependence of W59 fluorescence intensity, suggests the involvement of at least three different protonation states. Taken together, these data suggest different structural changes and thus different modes of cytochrome c binding at neutral and acidic pH. Differences between structural changes involved in the binding processes at acidic and neutral pH are also manifested in the corresponding UV−vis absorption spectra. At pH 6.5, the wavelength of the Soret band absorption maximum (λmax) (Figure S4) is substantially more blueshifted (from 412 to 404 nm) at the highest cardiolipin concentration than it is at pH 7.4 (412 to 409 nm). This observation suggests a significant population of high-spin states at the former pH.23 This shift is less pronounced at pH 6.7, where the Soret band blueshifts to 406 nm. The proposed population of high-spin states is corroborated by the appearance of a charge transfer band at 625 nm (CT2). As shown in Figure 1 and Figure S5, its intensity increases with increasing CL concentration at both acidic pH values. This increase is clearly more pronounced at pH 6.5. There is no CT2 band in the spectrum measured at pH 7.4.24 Concomitantly with the increase in CT2, we observed a decrease in the charge transfer band at 695 nm (CT1), which is an indicator of the native state with M80 as heme ligand (Figure 1 and Figure S5).31 The thus indicated change of the spin state at acidic pH is reminiscent of the behavior of denatured cytochrome c at acidic pH and is therefore indicative of the protonation of a histidine ligand as the probable cause.26 Hence our data strongly suggest that histidine is the sixth ligand in the previously reported fluorescing, partially unfolded low-spin liposome-bound protein.24,36 This notion is strongly corroborated by the results of a recent resonance Raman study of complexes formed between oxidized cytochrome c and cardiolipin dissolved in water.37 The current literature suggests that the electrostatic A and L sites of the protein are involved in its binding to CL-containing liposomes between pH 6 and 7.13,15,16 Furthermore, we have to consider recently investigated binding processes at neutral pH as being clearly distinct from A-site binding owing to their much lower binding affinity. To check whether the herein

Figure 2. Resonance Raman spectrum of a pH 6.5 50 μM ferricytochrome c solution at a cardiolipin concentration of 0 μM (red) and 1500 μM (gray) as obtained with 442 nm excitation, shown along with the decomposition (dashed black) and the total fit (solid black) of the latter spectrum. Sub-bands are labeled according to the notation of Abe et al.40

Each spin marker band has at least three components, indicative of the coexistence of at least three species. For the ν3 band, the specific sub-bands at 1485 and 1492 cm−1 are dominant; the two sub-bands above 1500 cm−1 contribute very little to the band profile. By taking into account the lower resonance enhancement expected for the high-spin species in comparison with their low-spin counterparts due to the blue shift of the Soret maximum, we estimated that only ∼10% of the proteins was still in a low-spin configuration. On the basis of the assignments reported by Oellerich et al.,9 the sub-bands 1995

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998

Letter

The Journal of Physical Chemistry Letters at 1485 and 1492 cm−1 are indicative of pentacoordinated and water-ligated hexacoordinated high-spin species, respectively. These assignments are further corroborated by the appearance of ν2 sub-bands at 1569 and 1577 cm−1, respectively, and a ν10 sub-band at 1625 cm−1. The low-spin sub-bands reflect some residual deprotonated bis-His species. The bis-His assignment is corroborated by the observed ν4, ν3, and ν10 components found at 1373, 1502, and 1639 cm−1, respectively.9 It is of note that we observed a weak “ν3” sub-band at 1478 cm−1 for which no current assignment is available in the literature. The RR spectra of ferricytochrome c at a high CL concentration and 150 mM NaCl in the absence and presence of ferrocyanide are shown in Figure S8. In the absence of ferrocyanide, the spectrum resembles that of a native (or nativelike) ferricytochrome c system. Upon the addition of ferrocyanide, the spectrum reveals complete photoreduction, which indicates that in the presence of salt ferricytochrome c exists in its native conformation even at high CL concentrations. This observation corroborates the notion that the binding observed at pH 6.5 is entirely electrostatic in nature. Taken together, our data suggest a reversible electrostatically driven binding of ferricytochrome c to CL membranes at mildly acidic pH that is reminiscent of the previously proposed L-site binding mode.16 Besides corroborating this binding process, our absorption and Raman data allow for an identification of the spin and ligation states of the corresponding liposomebound proteins. The combined data presented in this study suggest the following minimal reaction scheme (Scheme 1).

coexist. The role of H26 is likely facilitated by a breakage of the H26−P44 hydrogen bond in the f state.42 In summary, our results show that upon lowering the pH from 7.4 to 6.5 the binding of cytochrome c switches from partial to total electrostatic binding. This change is reminiscent of the L-site binding involving the lysine residues K22, K25, and K27 discovered by Nantes and coworkers.16,29,43 The role of A-site binding can be ruled out because the apparent ligand affinities indicated by our spectroscopic response data (Figure 1) are orders of magnitude lower than those reported by Kinnunen and coworkers.15,44 The observed change of the concomitant spin and ligation state suggest that the heme group of non-native conformations of oxidized cytochrome c adopted at high CL concentration and neutral pH is axially ligated by two histidines. Our resonance Raman data reveal coexisting hexa- and pentacoordinated high-spin states. Obviously, one expects that particularly pentacoordinated high-spin cytochrome c has a much higher capability of functioning as a peroxidase than corresponding misligated lowspin proteins.45 It is very likely that even minor pH variations in the intermembrane space around pH 7 could produce minor fractions of this species via L-site binding, which would then trigger an irreversible biochemical cascade that eventually facilitates the involvement of the protein in the apoptopic process. Full future characterization of this pentacoordinated high-spin species, along with measurements of its peroxidase activity, will be of paramount importance in the proper understanding of its influence on the pro-apoptotic activity of cytochrome c.



Scheme 1. Proposed CL−Cyt c Binding Process as the pH Changes

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b00597. Detailed experimental procedures (PDF)



AUTHOR INFORMATION

Corresponding Author

*Phone: 218-895-2268. E-mail: [email protected]. ORCID

Reinhard Schweitzer-Stenner: 0000-0001-5616-0722 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We acknowledge the Department of Chemistry at Drexel University for the partial funding of this work.

At neutral pH, liposome-bound proteins partition into native like (nf) and non-native-like ( f) conformations. Lowering the pH changes the mode of binding with either reduced overall binding affinity or a shift of the equilibrium between nf and f toward the former. Upon lowering the pH further the sixth ligand is protonated. As a consequence, the iron spin state of f switches from low to high spin. In principle, there are two candidate for the second histidine ligation, namely, H33 and H26. One would be tempted to opt for H33 as the most likely sixth ligand in the low-spin f state because the pK value of H26 lies at much lower values in the native state.41 However, the recent resonance Raman study of Milazzo et al. actually showed that both H33 and H26 can form bis-His complexes with H18 if one of the two is replaced by another amino acid.37 This suggests the coexistence of isomers, each exhibiting one of these two amino acid residues as the sixth ligand, similar to the isomers of oxidized alkaline cytochrome c where protein conformations with different lysine ligands of the heme iron

REFERENCES

(1) Therien, M. J.; Bowler, B. E.; Selman, M. A.; Gray, H. B.; Chang, I.-J.; Winkler, J. R. Long-Range Electron Transfer in Heme Proteins. Adv. Chem. Ser. 1991, 228, 191−199. (2) Green, D. R.; Kroemer, G. The Pathology of Mitochondrial Cell Death. Science 2004, 305, 626−629. (3) Kagan, V. E.; Tyurin, V. A.; Jiang, J.; Tyurina, V. A.; Ritov, V. B.; Amoscato, A.; Osipov, A. N.; Belikova, N. A.; Kapralov, A. A.; Kini, V.; Vlasova, I. I.; Zhao, Q.; Zou, M.; Di, P.; Svistunenko, D. A.; Kurnikov, I. V.; Borisenko, G. G. Cytochrome c Acts as a Cardiolipin Oxygenase Required for Release of Proapoptic Factors. Nat. Chem. Biol. 2005, 1, 223−232. (4) Belikova, N. A.; Vladimirov, Y. A.; Osipov, A. N.; Kapralov, A. A.; Tyurin, V. A.; Potapovich, M. V.; Basova, L. V.; Peterson, J.; Kurnikov, I. V.; Kagan, V. E. Peroxidase Activity and Structural Transitions of

1996

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998

Letter

The Journal of Physical Chemistry Letters Cytochrome c Bound to Cardiolipin-containing Membranes. Biochemistry 2006, 45, 4998−5009. (5) Hong, Y.; Muenzner, J.; Grimm, S. K.; Pletneva, E. V. Origin of the Conformational Heterogeneity of Cardiolipin-Bound Cytochrome C. J. Am. Chem. Soc. 2012, 134, 18713−18723. (6) Muenzner, J.; Toffey, J. R.; Hong, Y.; Pletneva, E. V. Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c. J. Phys. Chem. B 2013, 117, 12878−12886. (7) Sinibaldi, F.; Howes, B. D.; Piro, M. C.; Polticelli, F.; Bombelli, C.; Ferri, T.; Coletta, M.; Smulevich, G.; Santucci, R. Extended Cardiolipin Anchorage to Cytochrmoe c: a Model for ProteinMitochondrial Membrane Binding. JBIC, J. Biol. Inorg. Chem. 2010, 15, 689−700. (8) Sinibaldi, F.; Fiorucci, L.; Patriarca, A.; Lauceri, R.; Ferri, T.; Coletta, M.; Santucci, R. Insights into Cytochrome c - Cardiolipin Interaction. Role Played by Ionic Strength. Biochemistry 2008, 47, 6928−6935. (9) Oellerich, S.; Lecomte, S.; Paternostre, M.; Heimburg, T.; Hildebrandt, P. Peripheral and Integral Binding of Cytochrome c to Phospholipids Vesicles. J. Phys. Chem. B 2004, 108, 3871−3878. (10) Oellerich, S.; Wackerbarth, H.; Hildebrandt, P. Spectroscopic Characterization of Nonnative Conformational States of Cytochrome c. J. Phys. Chem. B 2002, 106, 6566−6580. (11) Oellerich, S.; Wackerbarth, H.; Hildebrandt, P. Conformational Equilibria and Dynamics of Cytochrome c Induced by Binding of Sodium Dodecyl Sulfate Monomers and Micelles. Eur. Biophys. J. 2003, 32, 599−613. (12) Sinibaldi, F.; Milazzo, L.; Howes, B. D.; Piro, M. C.; Fiorucci, L.; Polticelli, F.; Ascenzi, P.; Coletta, M.; Smulevich, G.; Santucci, R. The Key Role played by Charge in the Interaction of Cytochrome c with Cardiolipin. JBIC, J. Biol. Inorg. Chem. 2017, 22, 19−29. (13) Rytöman, M.; Mustonen, P.; Kinnunen, P. K. J. Reversible, Nonionic and pH-dependent Association of Cytochrome c with Cardiolipin- Phosphatidylcholine Liposomes. J. Biol. Chem. 1992, 267, 22243−22248. (14) Sinibaldi, F.; Howes, B. D.; Droghetti, E.; Polticelli, F.; Piro, M. C.; Di Pierro, D.; Fiorucci, L.; Coletta, M.; Smulevich, G.; Santucci, R. Role of Lysines in the Cytochrome c - Cardiolipin Interactions. Biochemistry 2013, 52, 4578−4588. (15) Rytöman, M.; Kinnunen, P. K. J. Evidence for two Distinct Acidic Phospholipid-binding Sites in Cytochrome c. J. Biol. Chem. 1994, 269, 1770−1774. (16) Kawai, C.; Prado, F. M.; Nunes, G. L. C.; Di Mascio, P.; Carmona-Ribeiro, A. M.; Nantes, I. L. pH-Dependent Interaction of Cytochrome c with Mitochondrial Mimetic Membranes: The Role of an Array of Positively Charged Amino Acid Residues. J. Biol. Chem. 2005, 280, 34709−34717. (17) Banci, L.; Bertini, I.; Reddig, T.; Turano, P. Monitoring the Conformational Flexibility of Cytochrome c at Low Ionic Strength by H-NMR Spectroscopy. Eur. J. Biochem. 1998, 256, 271−278. (18) Tuominen, E. K. J.; Wallace, C. J. A.; Kinnunen, P. K. J. Phospholipid-Cytochrome c Interaction. J. Biol. Chem. 2002, 277, 8822. (19) Kalanxhi, E.; Wallace, C. J. A. Cytochrome c Impaled: Investigation of the Extended Lipid Anchorage of a Soluble Protein to Mitochondrial Membrane Models. Biochem. J. 2007, 407, 179−187. (20) Rajagopal, K.; Schneider, J. P. Self-Assembling Peptides and Proteins for Nanotechnological Applications. Curr. Opin. Struct. Biol. 2004, 14, 480−486. (21) McClelland, L. J.; Mou, T.-C.; Jeakins-Cooley, M. E.; Sprang, S. R.; Bowler, B. E. Structure of a Mitochondrial Cytochrome c Conformer Competent for Peroxidase Activity. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 6648−6653. (22) Hanske, J.; Toffey, J. R.; Morenz, A. M.; Bonilla, A. J.; Schiavoni, K. H.; Pletneva, E. V. Conformational Properties of Cardiolipin-Bound Cytochrome c. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (1), 125−30. (23) Pandiscia, L. A.; Schweitzer-Stenner, R. Coexistence of Nativelike and Non-native Partially Unfolded Ferricytochrome c on the

Surface of Cardiolipin-Containing Liposomes. J. Phys. Chem. B 2015, 119, 1334−1349. (24) Pandiscia, L. A.; Schweitzer-Stenner, R. Coexistence of NativeLike and Non-Native Cytochrome c on Anionic lipsomes with Different Cardiolipin Content. J. Phys. Chem. B 2015, 119, 12846− 12859. (25) Bradley, J. M.; Silkstone, G.; Wilson, M. H.; Cheesman, M. R.; Butt, J. M. Probing a Complex of Cytochrome c and Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis. J. Am. Chem. Soc. 2011, 133, 19676−19679. (26) Elöve, G. A.; Bhuyan, A. K.; Roder, H. Kinetic Mechanism of Cytochrome c Folding: Involvement of the Heme and Its Ligands. Biochemistry 1994, 33, 6925−6935. (27) Takahashi, S.; Yeh, S.-R.; Das, T. K.; Chan, C.-K.; Gottfried, D. S.; Rousseau, D. L. Folding of Cytochrome c Initiated by Submillisecond Mixing. Nat. Struct. Biol. 1997, 4, 44−50. (28) Yeh, S.-R.; Takahashi, S.; Fan, B.; Rousseau, D. L. Ligand Exchange during Cytochrome c Folding. Nat. Struct. Biol. 1997, 4, 51− 56. (29) Kawai, C.; Pessoto, f. S.; Rodrigues, T.; Mugnol, K. C. U.; Tortora, v.; Castro, L.; Milicchio, V. A.; Tersariol, I. L. S.; Di Mascio, P.; Radi, R.; Carmona-Ribeiro, A. M.; Nantes, I. L. pH-Sensitive Binding of Cytochrome c to the Innert Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis. Biochemistry 2009, 48, 8335−8342. (30) Tsong, T. Y. The Trp-59 Fluorescence of Ferricytochrme c as Sensitive Measure of the Over-all Protein Conformation. J. Biol. Chem. 1974, 249, 1988−1990. (31) Schejter, A.; George, P. The 695 mμ Band of Ferricytochrome c and Its Relationship to Protein Conformation. Biochemistry 1964, 3, 1045−1049. (32) Taler, G.; Schejter, A.; Navon, G.; Vig, I.; Margoliash, E. The Nature of the Thermal Equilibrium Affecting the Iron Coordination of Ferric Cytochrome c. Biochemistry 1995, 34, 14209−14212. (33) Dragomir, I.; Hagarman, A.; Wallace, C.; Schweitzer-Stenner, R. Optical Band Splitting and Electronic Perturbations of the Heme Chromophore in Cytochrome c at Room Temperature Probed by Visible Electronic Circular Dichroism Spectroscopy. Biophys. J. 2007, 92, 989−998. (34) Pielak, G. J.; Oikawa, K.; Mauk, A. G.; Smith, M.; Kay, C. M. Elimination of the Negative Soret Cotton Effect of Cytochrome c by Replacement of the Invariant Phenylalanine Using Site-Directed Mutagenesis. J. Am. Chem. Soc. 1986, 108, 2724−2727. (35) Hagarman, A.; Wallace, C. J. A.; Laberge, M. M.; SchweitzerStenner, R. Out-of-plane Deformations of the Heme Group in Different Ferrocytochrome c Proteins Probed by Resonance Raman spectroscopy. J. Raman Spectrosc. 2008, 39, 1848−1858. (36) Serpas, L.; Milorey, B.; Pandiscia, L. A.; Addison, A. W.; Schweitzer-Stenner, R. Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes. J. Phys. Chem. B 2016, 120, 12219−12231. (37) Milazzo, L.; Tognaccini, L.; Howes, B. D.; Sinibaldi, F.; Piro, M. C.; Fittipaldi, M.; Baratto, M. C.; Pogni, R.; Santucci, R.; Smulevich, G. Unravelling the Non-Native Low-Spin State of the Cytochrome c− Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only. Biochemistry 2017, 56, 1887−1898. (38) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. Complete Assignment of Cytochrome c Resonance Raman Spectra via Enzymatic Reconstitution with Isotopically Labeled Hemes. J. Am. Chem. Soc. 1993, 115, 12446−12458. (39) Malyshka, D.; Schweitzer-Stenner, R. Ferrocyanide-Mediated Photoreduction of Ferricytochrome c Utilized to Selectively Probe Non-native Conformations Induced by Binding to CardiolipinContaining Liposomes. Chem. - Eur. J. 2017, 23, 1151−1156. (40) Abe, M.; Kitagawa, T.; Kyogoku, Y. Resonance Raman Spectra in Octaethylporphyrin-Ni(II) and Mesodeuterated and 15N Substituted Derivatives. II. A Normal Coordinate Analysis. J. Chem. Phys. 1978, 69, 4526−4534. 1997

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998

Letter

The Journal of Physical Chemistry Letters (41) Cohen, D. S.; Pielak, G. J. Stability of Yeast Iso-1 Ferricytochrome c as a Function of pH and Temperature. Protein Sci. 1994, 3, 1253−1280. (42) Balakrishnan, G.; Hu, Y.; Spiro, T. G. H26 Protonation in Cytochrome c Triggers Microsecond β-sheet Formation and Heme Exposure: Implications for Apoptosis. J. Am. Chem. Soc. 2012, 134, 19061−19069. (43) Kawai, C.; Ferreira, J. C.; Baptista, M. S.; Nantes, I. L. Not Only Oxidation of Cardiolipin Affects the Affinity of Cytochrome c for Lipid Bilayers. J. Phys. Chem. B 2014, 118, 11863−11872. (44) Gorbenko, G. P.; Molotkovsky, J. G.; Kinnunen, P. K. J. Cytochrome c Interaction with Cardiolipin/Phosphatidylcholine Model Membranes: Effect of Cardiolipin Protonation. Biophys. J. 2006, 90, 4093. (45) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Crystal Structure Determination of Classical Horseradish Peroxidase at 2.15 Å Resolution. Nat. Struct. Biol. 1997, 4, 1032−1038.

1998

DOI: 10.1021/acs.jpclett.7b00597 J. Phys. Chem. Lett. 2017, 8, 1993−1998