Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants

Structural and functional insights into lysine acetylation of cytochrome cusing mimetic point mutants Inmaculada M´ arquez 1 , Gonzalo P´ erez-Mejı´as 1 , Alejandra Guerra-Castellano 1 , Jos´ e Luis Olloqui-Sariego 2 , Rafael Andreu 2 , Juan Jos´ e Calvente 2 , Miguel A. De la Rosa 1 and Irene Dı´az-Moreno 1 1 Institute for Chemical Research (IIQ), Scientific Research Centre Isla de la Cartuja (cicCartuja), University of Seville –CSIC, Spain 2 Departament of Physical Chemistry, University of Seville, Spain Keywords acetylation; bioenergetics; cytochrome c; electron transport chain; post-translational modifications Correspondence I. Dı´az-Moreno, Institute for Chemical Research (IIQ), Scientific Research Centre Isla de la Cartuja (cicCartuja), University of Seville –CSIC, Avda. Am´ erico Vespucio 49, Sevilla 41092, Spain Tel: +34 954 489 513 E-mail: [email protected] Gonzalo P´ erez-Mejı´as and Alejandra Guerra- Castellano contributed equally to the article (Received 1 June 2021, revised 24 August 2021, accepted 27 August 2021) doi:10.1002/2211-5463.13284 Abstract Post-translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome cand its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylation-induced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome cwith glutamine to mimic lysine acetylation and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl-mimetic mutants, but their thermal stability was significantly altered. NMR chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome cto its physiological electron donor, cytochrome c 1 —which is a component of mitochondrial complex III, or cytochrome bc 1 —thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome coxidase. Abbreviations A, pre-exponential factor; AcK, acetyl-lysine; ANOVA, one-way analysis of variance; BLI, Bio-layer interferometry; Cc, cytochrome c;Cc 1 , cytochrome c 1 ;CcO, cytochrome coxidase; CIII, complex III; CIV, complex IV; CSP, chemical shift perturbation; DMEM, Dulbecco’s modified Eagle’s medium; E. coli,Escherichia coli;E 0 , midpoint reduction potential; E 1/2 , midpoint redox potential value; ETC, electron transport chain; HSQC, heteronuclear single quantum correlation; ITC, isothermal titration calorimetry; K D , dissociation equilibrium constant; k s , electron transfer rate constant; LB, luria-Bertani; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MD, molecular dynamics; MEF, mouse embryonic fibroblast; MOA, 8-Mercaptooctanoic acid; MOOL, 8-Mercapto-1-octanol; NHE, normal hydrogen electrode; NOESY, nuclear Overhauser effect spectroscopy; P1, first principal component; PCA, principal component analysis; PDB, protein data bank; PMSF, phenylmethanesulfonyl fluoride; PTMs, post-translational modifications; RG, radius of gyration; RMSF, root mean square fluctuation; RSA, rabbit serum albumin; SAMs, self-assembled monolayers; SDS/PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; T, temperature; T m , midpoint melting temperature; TOCSY, total correlation spectroscopy; UV, ultraviolet; Vis, visible; WT, wild-type; ε, extinction coefficient. 1FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Post-translational modifications (PTMs) have traditionally been associated with mechanisms that cells use to regulate and expand the functionality of their biomolecules [1–3]. These modifications can be reversible or irreversible and can include covalent addition of complex molecules, chemical groups, and proteins/peptides, as well as protein cleavage and amino acid modification. Recent studies however have shown that certain PTMs (including acetylation, glycosylation, methylation, nitration, S-palmitoylation, phosphorylation, S-nitrosylation, sumoylation, and ubiquitination) or the loss of modification sites in proteins are responsible for the development of certain diseases [4–7]. Based on the PTM database (PTMD), phosphorylation is the most frequent PTM associated with human disease (http://ptmd.biocuckoo.org/)[6]. These data are striking because acetylation is the major covalent modification to occur in eukaryotic cells, where over 60% of mitochondrial proteins (soluble and membrane-embedded) have one or more acetylation sites [8–11]. Acetylation actually regulates diverse protein properties, including DNA interaction, subcellular localization, transcriptional activity, and stability [12]. Acetylation is a reversible enzymatic process carried out by lysine acetyltransferases, in which the ε-amino group of lysine residues is reversibly modified by the acetyl group. Interestingly, the acetylation process can take place by means of a non-enzymatic mechanism in the mitochondrial matrix, where the addition of the acetyl group is promoted by high concentrations of acetyl-CoA and the alkaline pH [13]. A principal mitochondrial protein affected by PTMs is respiratory cytochrome c(Cc), which has also been described as a target for lysine acetylation [14]. Under homeostasis, Ccis known to be located in the mitochondrial intermembrane space and acts as an electron carrier between complex III (CIII) and complex IV (CIV) in the electron transport chain (ETC). It was recently reported that CIII and CIV host two Ccbinding sites each: the so-called proximal, or catalytic site, which is the relevant functional position for electron transfer, and the distal, or non-productive site, which facilitates the turnover and sliding of Ccmolecules [15,16]. In the case of the respiratory supercomplexes, the close arrangement of CIII and CIV suggests a ‘restrained diffusion pathway’ for Ccmolecules between the two complexes [17]. Nevertheless, several internal and external cell stimuli can induce the translocation of Ccto other cell compartments (nucleus, vacuoles, zymogen granules, cytosol, and even the rough endoplasmic reticulum), where Ccbehaves as a moonlighting protein able to interact with an ample set of non-redox proteins [18–28]. As mentioned above, Cccan be acetylated on its lysine residues [29]. Several studies with chemically acetylated hemeprotein showed that lysine acetylation modulates respiratory chain functioning by inducing a substantial drop in the Ccreduction/oxidation rate [29] and, consequently, a decrease in Cc-mediated electron transfer [30]. Notably, these in vitro acetylation assays simultaneously affect multiple lysines, being difficult to assign any acetylation effect to any particular lysine. Downregulation of mitochondrial electron flow promotes inhibition of cellular respiration, also known as the Warburg effect [31]. So far, in vivo acetylation of both Lys8 and Lys53 has only been described in mammalian Cc. Acetylation of Lys8 was observed in a proteomic analysis of mouse liver mitochondria, whereas acetylation of Lys53 was identified in prostatic cancer cells [8,32]. This association with prostate cancer led to acetylation of Lys53 being the first to become the object of study [32]. The interaction between Ccand its partners depends strongly on the ionic strength of the media, revealing the importance of charged residues such as lysines in its associations [33]. Lys8 and Lys53, with their side chains exposed to the solvent, are involved in the binding of Ccto most of its metabolic partners as they contribute to a positively charged Lys-rich patch at the hemeprotein surface [16,22,34–37]. Since acetylation neutralizes the positive charge of lysines, this PTM might induce drastic changes in the physicochemical and structural features of Ccand could therefore alter interactions with its metabolic partners. The difficulty of preserving the acetylation state of Ccand the low yield obtained from cell extracts make it challenging to analyze. Furthermore, the specific acetyltransferase acting on the hemeprotein remains unknown. Hence, recourse was made to amino acid point mutations to mimic the PTM. The Lys-to-Gln mutation can mimic the acetylation-dependent neutralization of the positive lysine charge, so that the resulting Ccspecies could be used as acetyl-mimetic mutants. As part of a strategy to gain insight into the structural and functional analysis of lysine-acetylated Cc, we here characterize two acetyl-mimetic mutants of Cc, namely K8Q and K53Q Cc, using K8A and K53A mutants as controls. Methods Site-directed mutagenesis, protein expression, and purification of recombinant proteins Site-directed mutagenesis was performed on a pBTR1 plasmid comprising the CYCS gene coding for human Cc, 2FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. along with the CYC3 gene of the yeast Ccheme lyase [38]. The CYC3 gene is required for the proper cytoplasmic maturation of Cc, as the heme lyase covalently attaches the heme group to the apoprotein of Cc. Primers used for mutagenic PCR are detailed in Table S1. The expression of wild-type (WT) and acetyl-mimetic Cc species was performed as previously described [39].Briefly, Escherichia coli (E. coli) BL21 (DE3) cells were transformed with either pBTR1-CcWT or pBTR1-Ccacetyl-mimetic plasmids. The cells were cultured at 30 °C with agitation (150 rpm) in Luria-Bertani (LB) medium supplemented with 100 μgmL −1 ampicillin for 20 h. Cells were harvested by centrifugation (9000 gfor 10 min), suspended in lysis buffer (10 mMTricine-NaOH, pH 8.5, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02 mgmL −1 DNase and complete protease inhibitor) and physically ruptured by sonication. Cellular debris was separated by centrifugation at 14 000 gfor 30 min at 4 °C. For the NMR experiments, 15 N-labeled protein was expressed in minimal M9 medium supplemented with 16.7 μgmL −1 δ-aminolevulinic acid hydrochloride and 15 NH 4 Cl as sole nitrogen source. Protein purification was carried out by ion chromatography in a Nuvia-S (Bio-Rad, Hercules, CA, USA) column using a FPLC system (Bio-Rad). The purity of protein samples was routinely tested by electrophoretic (Fig. S1A) and spectrophotometric analyses (see below). In all cases, the A 280 /A 550 ratio of the resulting Ccpreparations in the reduced state was about 1.1— as previously reported to be in pure Ccsamples [39]—with a maximum 10% deviation. Immunoblot was performed with rabbit anti-human Ccserum (obtained by immunizing male rabbits with full-length recombinant Ccsuspended in a 0.85% NaCl solution) as primary antibody. HRP-conjugated secondary antibody was used for detection (Sigma-Aldrich Co., St. Louis, MO, USA, catalog number A0545). The immunoreactive bands were detected using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Chalfont St. Giles, UK). The molecular masses of Ccvariants were checked by running a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Tryptic digestion analysis confirmed the point mutations of the Ccspecies. Protein concentration was determined by visible (Vis) spectrophotometry, using an extinction coefficient (ε 550nm )of28.92mM −1 cm −1 for reduced Cc[39]. Protein samples were dialyzed against 10 mMsodium phosphate buffer pH 5.5 or pH 7.4 for NMR, or CD and bio-layer interferometry (BLI) experiments. The C10A mutant of Arabipdosis thaliana cytochrome c 1 (Cc 1 ), used to avoid protein dimerization, was expressed as previously described [15].E. coli BL21 (DE3) cells were co-transformed with pET-Cc 1 -C10A and pEC86—coding for the CYC3 gene of the yeast Ccheme lyase—to produce the soluble domain of Cc 1 . Cells were grown for 24 h at 150 rpm and 30 °C in LB-rich medium supplemented with 16.7 μgmL −1 δ-aminolevulinic acid hydrochloride, 50 μgmL −1 kanamycin, and 12 μgmL −1 chloramphenicol. Cells were collected by centrifugation (9000 gfor 10 min) and then suspended in lysis buffer (20 mMTris/HCl, pH 8.0, supplemented with 1 mMPMSF, 0.02 mgmL −1 DNase and complete protease inhibitor). The periplasmic fraction was obtained by the freeze-thaw lysis method. The resulting suspension was centrifuged at 20 000 gfor 30 min and loaded onto a Nuvia-Q column (Bio-Rad). Cc 1 was eluted with a NaCl linear gradient in 20 mMTris/HCl, pH 8.0. The fractions containing Cc 1 were further purified by FPLC using a gel filtration ENrich SEC 70 column (Bio- Rad). Protein concentration was determined by Vis spectrophotometry, using an extinction coefficient (ε 552nm )of 28 mM −1 cm −1 for reduced Cc 1 [15]. The recombinant protein was dialyzed against 10 mMsodium phosphate buffer pH 7.4 for BLI experiments. CD spectroscopy CD spectra were recorded using a Jasco ® J-815 spectropolarimeter equipped with a Peltier temperature control system. The secondary structure analyses were carried out by recording far-ultraviolet (UV) CD spectra (185–250 nm) at 25 °C in a 1-mm quartz cuvette. Samples contained 3 μM protein in 10 mMsodium phosphate (pH 7.4), supplemented with 10 μMpotassium ferricyanide. 20 scans were averaged for each sample. The coordination of the heme iron atom to the S δ atom of Met80 (the sixth axial ligand of heme group) was analyzed by visible (B-band) recording near-UV/Vis CD spectra (300–600 nm) in a 10 mm quartz cuvette at 25 °C. Samples contained 30 μMprotein in 10 mMsodium phosphate (pH 7.4), supplemented with 100 μMpotassium ferricyanide. Thermal unfolding was monitored between 10 °C and 105 °C (with a heating rate of 1 °Cmin −1 ) by recording the CD signal at far-UV in a 10 mm quartz cuvette. For these assays, the oxidized Ccspecies were at 3 μMfinal concentration in 10 mMsodium phosphate (pH 7.4), supplemented with 10 μMpotassium ferricyanide. The heme coordination of the Ccspecies was monitored from 10 °C and 105 °C (with a heating rate of 1 °Cmin −1 ) by recording near-UV/ Vis CD spectra from solutions in a 10 mm quartz cuvette. Samples contained 30 μMCcin 10 mMsodium phosphate buffer (pH 7.4), supplemented with 100 μMpotassium ferricyanide. The midpoint melting temperature (T m ) values were obtained from the fits of principal components obtained from CD and fluorescence spectra measured on temperature ramps. Electronic absorption spectroscopy The redox state reversibility of heme group was checked by recording absorption spectra from 350 to 600 nm after the addition of 10 equivalents of potassium ferricyanide as an 3FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c oxidizing agent and 50 equivalents of sodium ascorbate as a reducing agent. Spectra of 5 µMof Ccspecies were recorded at 25 °Cin10m Msodium phosphate buffer (pH 7.4). The coordination of the heme group of Ccwas analyzed by monitoring the absorption changes at 699 nm, indicative of the heme Fe-Met80(S δ ) bond as reported previously [40]. Electronic absorption spectra were recorded in the 600– 750 nm range at 25 °C, using a Jasco ® V-650 spectrophotometer in a 1-mL quartz cuvette with a path length of 10 mm. Samples contained 0.2 mMoxidized Ccin 10 mM sodium phosphate (pH 5.8), supplemented with 0.2 mM potassium ferricyanide. For pH titration studies, the pH of each sample was adjusted to the reported values by adding aliquots of 0.1–0.5 M NaOH or 0.1–0.5 M HCl. To calculate the pK a value of each protein, the absorbance changes at 699 nm were fitted to the Henderson-Hasselbalch equation expressed in the following terms [41]: Y¼Absmin þAbsmax Absmin 1þeðXpKaÞ=NS (1) where Y is the experimental value for absorbance at 699 nm, Abs max and Abs min are the maximum and minimum values for absorbance at 699 nm, X is the pH of the protein sample, pK a is the apparent pK a value for alkaline transition, and NS is the slope of the sigmoid. Molecular dynamics simulations The initial structures were based on the NMR structure of reduced human Cc(Protein Data Bank, PDB, ID: 2N9I; [42]). All reduced mutants (K8A, K8Q, K8AcK, K53A, K53Q, and K53AcK) structural models were built using the WT protein as a template with UCSF Chimera software [43]. The force field parameters for acetyl-lysine [44] and heme group [45] were used to generate the topology and coordinate files, required for the simulation, with TLEAP module of AMBER [46]. Molecular dynamic (MD) simulations were performed using the OpenMM [47] software in CUDA platform with AMBER 14SB force field [48] and particle mesh Ewald electrostatics with an Ewald summation cut-off of 10 ˚ A. For each simulation, the system was neutralized with chlorine counter-ions according to the total charge of the protein and solvated with optimal-3-charge, 4-point rigid water model water molecule [49]. The whole system was subjected to 2500 steps of energy minimization at 298 K. Langevin thermostat was used to control the temperature with a friction coefficient of 1 ps −1 and a step size of 0.002 ps. Each system was subjected to 900 ns MD simulation. For the trajectory analysis, the CPPTRAJ module of AMBER was used [50] to analyze the last 50 ns of simulation. NMR spectroscopy NMR experiments were performed on a 700 MHz Bruker Avance-III spectrometer equipped with a cryoprobe. Backbone amide group resonances of Ccvariants were monitored by recording 2D 15 N- 1 H heteronuclear single quantum correlation (HSQC) experiments. Samples contained 150 μMof reduced 15 N-labeled Ccin 20 mMcitrate buffer (pH 5.5), 5% D 2 O and sodium ascorbate to ensure the redox state of the sample. NMR assignments of the 15 N and 1 H nuclei of reduced WT Cc(BMRB accession number 25907) were taken from previous work [42]. Additional 3D 15 N- 1 H total correlation spectroscopy (TOCSY)- HSQC and 3D 15 N- 1 H nuclear Overhauser effect spectroscopy (NOESY)-HSQC experiments were recorded to identify the resonances of the Lys-Gln or Lys-Ala Cc mutants. The experimental data were processed using Bruker TopSpin NMR 4.1.1 software (Bruker), and chemical shift perturbations (CSPs) were analyzed with NMRFAM- Sparky NMR assignment tool [51]. Bio-layer interferometry measurements BLI measurements were performed on the Octet RED 96e platform (Fort´ eBio) using Ni-NTA (NTA) Biosensors (Fort´ eBio). The protocol used for the kinetic analysis is detailed in Table S2. The biosensors were hydrated in a 96-well black plate for 10 min with BLI buffer containing 10 mMsodium phosphate (pH 7.4) supplemented with 0.01 μMrabbit serum albumin (RSA) and 0.01% Triton X-100 as blocking agents to reduce non-specific binding to the biosensor. The biosensors were incubated with 200 μL of His 6 -tagged Cc 1 sample (50 μgmL −1 diluted in BLI buffer) at the loading step for immobilization of the analyte into the NTA matrix. Binding of WT and acetyl-mimetic Ccvariants was analyzed at ligand concentrations between 0.3215 and 10 μMdiluted in BLI buffer and performed in duplicate. The surface of the biosensor was regenerated by 10 mMglycine (pH 1.7) using the standard protocol described by the manufacturer. The experimental data were processed with Data Analysis HT 12.0 software (FORT ´ EBIO) and analyzed with Origin Lab Origin(Pro), Version 2019 (OriginLab Corporation, Northampton, MA, USA), using the non-linear equation described by M¨ uller-Esparza et al.[52]: Y¼Bmax X KDþXþNSX (2) where Y is the wavelength shift at equilibrium, B max is the maximum wavelength shift, X is the effector complex concentration, K D is the equilibrium dissociation constant, and NS is the slope of the linear components. 4FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. Isolation of mitochondria from mouse embryonic fibroblast cell lines Mouse embryonic fibroblast (MEF) cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO 2 . MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Co.) containing 4500 mgL −1 glucose (Sigma-Aldrich Co.), supplemented with 10% heatinactivated fetal bovine serum (Sigma-Aldrich Co.), 2 mM L-glutamine, 100 UmL −1 streptomycin, 100 μgmL −1 penicillin and 0.11 mgmL −1 sodium pyruvate (Sigma-Aldrich Co.). Cells were grown to 80% confluence in 24-well plates with 500 μL of DMEM. Mitochondria were obtained by differential centrifugation as reported previously [53]. Cytochrome c oxidase activity To assess the functionality of acetyl mimetics as electron donors, we tested their ability to reduce cytochrome coxidase (CcO) in a mitochondrial context using a CcO activity kit (Biochain, Newark, CA, USA), as reported previously [54]. Activity was measured on mitochondria isolated from MEF cell lines in a Jasco ® V-650 spectrophotometer in a 0.1-mL quartz cuvette with 10 mm path length, according to the manufacturer’s instructions. The Abs 550 slopes of at least three independent experiments were calculated and correlated with CcO activity values. Statistical analysis Data were analyzed using Origin Lab Origin(Pro), Version 2019 (OriginLab Corporation, Northampton, MA, USA). Data are expressed as means SD. One-way analysis of variance (ANOVA) was employed for comparisons of multiple groups. Electrochemical measurements Linear scan voltammetric measurements were performed with an AUTOLAB PGSTAT 30, from Eco Chemie B.V, in a three-electrode undivided glass cell, equipped with a gas inlet and thermostatted with a water jacket. Working electrodes were polycrystalline gold with a 0.0314 cm 2 geometric area. Prior to performing measurements, the gold surface was cleaned by successively polishing it with 0.3 and 0.05 µm alumina, rinsed with Millipore water, then sonicated in absolute ethanol to remove residual alumina. Then, the electrode surface was dried and chemically cleaned using ‘piranha’ solution (7 : 3 mixture of concentrated H 2 SO 4 (95%) and 30% v/v H 2 O 2 ). Gold electrodes were modified with thiol self-assembled monolayers (SAMs) by immersing the electrode in ethanol solutions containing 1m M8-mercaptooctanoic acid (MOA) and 2.5 mM8- mercapto-1-octanol (MOOL), from Sigma-Aldrich Co., for 30 min at room temperature. Protein immobilization was carried out by depositing on the modified electrode a 15 µL drop of a solution containing 20 µMprotein, 10 mMsodium phosphate buffer solution (pH 7) for 45 min at 4 °C. After protein incubation, the electrodes were rinsed with water and washed with the working buffer solution. The counter and reference electrodes were a Pt bar and an Ag/AgCl/ NaCl-saturated electrode, respectively. Reported potential values were corrected to the normal hydrogen electrode (NHE) potential scale by adding +192 mV to the experimental potential values. Working solutions contained 20 mMsodium phosphate buffer as supporting electrolyte, at pH 7.0 0.1. All measurements were performed under argon atmosphere. Results and Discussion Physicochemical features of cytochrome c are mainly unchanged in the two acetyl-mimetic mutants Cccontains 18 lysine residues but only Lys8 and Lys53 are acetylated in vivo [8,32]. Each residue is located in opposite regions of the hemeprotein (Fig. 1 A): Lys8 in helix I and Lys53 in helix II, with their respective side chains well exposed to the solvent. To explore how acetylation of Lys8 and Lys53 influences the physicochemical features of Cc, we designed two acetyl-mimetic mutants of Cc, replacing the corresponding lysines with glutamines (K8Q and K53Q Cc mutants) to resemble the change in charge that occurs when lysine becomes acetylated (Fig. 1B). To check the specific effect of these point mutations, Lys-to-Ala mutants were also studied (K8A and K53A, Fig. 1B). Since mutation of lysine to glutamine or alanine neutralizes the positive charge, we first calculated the surface electrostatic potential of the WT, point mutants (K8A, K8Q, K53A, and K53Q), and acetylated forms (K8AcK and K53AcK) of Ccat physiological ionic strength. In all cases, the lysine substitution resulted in a loss of positive potential at the surface area surrounding the mutated residue (Fig. 2), consistent with the charge difference between the ionic amino-terminal group of lysine in WT Cc and the neutral character of acetyl-lysine, glutamine, or alanine in the mutants. The similar surface electrostatic potentials of the acetyl-lysine variants (K8AcK and K53AcK) and the glutamine mutants (K8Q and K53Q) suggest that the Lys-to-Gln substitution could lead to good mimetics of acetylated Cc. Glutamine and alanine site-directed mutants were thus designed, expressed, and purified (see Methods section). The molecular mass of all purified mutants was corroborated by SDS/PAGE (Fig. S1A) and 5FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c MALDI-TOF mass spectrometry (Fig. 3A). The peak closest to 12 500 m/z in all mass spectra corresponds to the formation of a Schiff base adduct, as described previously [55]. Since the molecular masses of the glutamine mutants (K8Q and K53Q) were the same as that of the WT Cc, protein spots from SDS/PAGE Fig. 1. 3D structure of cytochrome cand formula of selected amino acids. (A) Ribbon representation of human Ccstructure (PDB, ID: 2N9I [42]), with the heme group (green color) and Lys8, Lys53, and Trp59 represented by sticks. The different foldon units are colored as follows: foldon I in blue, foldon II in green, foldon III in yellow, foldon IV in red, and foldon V in purple. (B) Chemical structure of acetyl-lysine, lysine, glutamine, and alanine. Fig. 2. Surface electrostatic potentials of WT and mutant species of cytochrome c. Electrostatic potentials were calculated at 150 mMionic strength. Green circles denote mutated residues at position 8 (upper) and 53 (lower). 6FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. bands were subjected to trypsin digestion to confirm the Lys-to-Gln substitution from the masses of the resulting fragments (Fig. S1B). UV-Vis absorption spectra clearly confirmed the typical hallmarks of ctype cytochromes in all mutants (Fig. 3B). The spectra for WT and mutant Ccin the reduced state show a sharp maximum at ca. 415 nm (Soret band), as well as two lower maximums at 520 and 550 nm (αand β bands, respectively) that merge in the oxidized state. The redox state reversibility of the heme group in all samples was checked by recovering original absorption spectra upon addition of potassium ferricyanide or sodium ascorbate as oxidizing or reducing agent, respectively. The effect of mutations on the secondary structure of Ccwas analyzed by far-UV CD spectroscopy of WT and mutant Ccspecies. The most prominent change was observed in the 195-nm band with the K8Q mutant, suggesting a substantial increase in αhelix content (Fig. 4). A detailed analysis using the CDPRO software package (Fig. S2) not only confirmed the increase in α-helix content in K8Q but also revealed a significant increase in K53Q. Since subtle changes in the heme group can alter the redox properties and physiological functions of Cc, the CD spectra in the visible region were also recorded to provide further information on the heme environment [56]. In its native form, the heme iron atom of Ccis hexacoordinated, with the fifth and sixth axial ligands being His18 and Met80, respectively [57]. The CD spectra of WT Ccand all mutants clearly exhibited the distinctive B-band splitting corresponding to the Fig. 3. Mass and visible absorption spectra of WT and mutant species of cytochrome c. (A) MALDI-TOF spectra of WT and Ccmutants. The molecular mass of WT, K8Q, and K53Q Ccis 12 235 Da, whereas that of K8A and K53A Ccis 12 179 Da. (B) Visible absorption spectra of oxidized (dashed line) and reduced (solid line) Ccin the presence of potassium ferricyanide and sodium ascorbate, respectively. The color code is the same as in panel (A). a.u., arbitrary units. 7FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c Cotton effect in the Soret absorption region [58]. Taken together, these findings confirm that Lys8 and Lys53 mutations have a negligible effect on the heme environment. Since the stability of the heme crevice is preserved under physiological conditions, the effect of pH on the heme environment was checked by Vis absorption spectroscopy. In the pH range of 1–12, at least five different conformations of Cchave been described as a result of changes in protein folding and/or heme axial coordination [59]. The transition from state III to state IV, called alkaline transition, is induced by an increase in pH [60]. This state transformation involves replacing the axial ligand Met80 with a lysine residue (Lys72, Lys73, or Lys79) [61,62]. The interaction between heme iron and Met80 in the oxidized form of Cccauses a maximum charge-transfer peak in the absorption spectra at 699 nm [40,63], which disappears as the alkaline transition occurs (Fig. S3,left panels). The presence of the 699 nm maximum at the initial pH value (pH ca. 6.3) indicates the integrity of protein folding [64]. The intensity of this charge-transfer peak was recorded in the pH 6.0–12.5 range, and the fitting of titration curves (Fig. S3,right panels) provided the pK a values for the alkaline transition of all mutants included in this study. Considering that Tyr48- phosphomimetic Ccmutants have lower but biologically relevant pK a transition values [40,65], one would expect that the alkaline transition of mutants at Lys53, which is in the same loop as Tyr48, would also be altered. However, the pK a values of all mutants were very close to that of WT Cc(Table 1). These results suggest that the native heme environment is preserved in acetyl-mimetic Ccspecies, consistent with exposure Fig. 4. Analysis of the secondary structure and heme environment of the WT and mutant species of cytochrome c. (A) Far-UV and (B) near- UV/Vis CD spectra of oxidized WT, K8A, K8Q, K53A, and K53Q Ccat 3 and 30 µM, respectively. 8FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. of the side chains of lysine, alanine and glutamine residues at positions 8 and 53 to solvent without altering the hydrogen bond pattern around the heme crevice [40]. Conservation of Met80-Fe bond distance, as predicted by theoretical MD calculations, also confirms the preservation of heme moiety (Fig. S4). Thermal stability of cytochrome c is differentially modified depending on the mutated residue The conformational stability of Ccdepends greatly on the protein environment, as well as the residues surrounding the heme group [66]. Changes in H-bond networks, hydrophobic interactions, steric restrictions, and even the protein sequence can alter the T m of the protein. To explore the effect of temperature on the secondary structure and heme moiety of Ccspecies, thermal stability assays were monitored by recording the CD and fluorescence spectra in the far-UV and near-UV/Vis ranges, respectively, in the temperature interval 10–105 °C. To ensure the preservation of Fe- Met80 coordination, the experiments were carried out at pH 7.4. The complex behavior of CD and fluorescence spectra required principal component analysis (PCA) in which the first principal component (P1) of spectra was fitted to a three-state or four-state model, depending on the assay (Fig. S5). The fitting of P1 provided T m values for the different thermal transitions that the mutants underwent along the temperature ramp (Table 2). The thermal unfolding profile from the representation of P1 and temperature in the far-UV CD spectra of all mutants showed similar thermal behavior to the WT protein and consisted of two thermal transitions (Fig. S5). These thermal unfolding profiles have been explained using a previous folding model, based on the division of Ccinto five folding regions (Fig. 1) with different thermal stabilities [67]. According to the model, the most stable region included helix I and helix IV (foldon I), while the Ω-loop between Thr19 and Phe36 residues and helix III comprised the second folding unit (foldon II). Denaturation of these two folding units may correspond to the second thermal transition observed in the far-UV CD spectra, since foldons I and II are helix-rich regions. The following region (foldon III) corresponds to two short stretches of residues from Phe36 to Lys39 and Gly56 to Gly60, while foldon IV includes the loop extending between helix III and helix IV and contains Met80 residue. The most unstable region (foldon V) extends from Thr40 to Lys55, between the two stretches of foldon III. The configuration of these three last regions is less structured and may thus be responsible for the first thermal transition. Since the Lys8 and Lys53 residues are located in helix I in foldon I and helix II in foldon V, respectively, preservation of the Ccthermal profile in the far-UV range indicates that the mutation of these residues does not alter foldon stability. The T m values for WT and mutant Ccspecies were quite similar (Table 2), with small differences consistent with the minor changes in secondary structure previously described in Figs 4and S2. However, the P1 plot of the near-UV/Vis CD spectra showed that the Lys8 mutations, especially in the K8A mutant, considerably modified the thermal behavior of Cc. While structural features of the heme moiety, such as heme plane bending, aromatic residue packing, and heme internal electric field [68,69], determine the visible B-band spectra, the thermal profile variations in the near-UV/Vis CD region could correspond to conformational changes in the heme crevice. The appearance of a new thermal transition at ca. 28 °C indicates the influence of the Lys8 mutation on the heme moiety (Fig. S5, Table 2). This residue is Table 1. pK a values for alkaline transition in oxidized WT and mutant cytochrome c. pK a WT 9.48 0.05 K8A 9.54 0.03 K8Q 9.56 0.06 K53A 9.42 0.04 K53Q 9.31 0.03 Table 2. Midpoint melting temperature (T m ) values for oxidized WT and mutant cytochrome cas calculated within the 185–250 nm (UV) and 300–600 nm (Vis) ranges by CD and fluorescence spectroscopy. T m (°C) CD spectra Fluorescence 185–250 nm 300–600 nm 185–250 nm WT 55.5 0.8/ 87.1 0.2 62.4 1.1/ 87.4 0.3 48.6 2.4/ 58.2 0.9/ 93.4 1.6 K8A 52.5 0.2/ 86.5 0.2 28.5 0.8/ 56.1 0.6/ 84.1 1.2 53.2 0.4/ 88.4 0.4 K8Q 53.2 0.3/ 88.2 0.4 29.1 0.3/ 69.1 3.7/ 87.8 0.7 51.2 0.3/ 90.1 0.3 K53A 57.7 4.6/ 82.1 1.7 49.0 0.8/ 83.5 0.2 52.4 0.6/ 88.8 2.5 K53Q 55.7 0.4/ 87.8 0.3 53.6 0.5/ 87.0 0.6 57.6 0.5/ 92.5 0.3 9FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c Rosa MA and Dı´az-Quintana A (2015) Respiratory complexes III and IV can each bind two molecules of cytochrome cat low ionic strength. FEBS Lett 589, 476–483. 17 P´ erez-Mejı´as G, Guerra-Castellano A, Dı´az-Quintana A, De la Rosa MA and Dı´az-Moreno I (2019) Cytochrome c: surfing off of the mitochondrial membrane on the tops of complexes III and IV. Comput Struct Biotechnol J17, 654–660. 18 Soltys BJ, Andrews DW, Jemmerson R and Gupta RS (2001) Cytochrome-clocalizes in secretory granules in pancreas and anterior pituitary. Cell Biol Int 25, 331– 338. 19 Martı´nez-F´ abregas J, Dı´az-Moreno I, Gonz´ alez-Arzola K, Janocha S, Navarro JA, Herv´ as M, Bernhardt R, Dı´az-Quintana A and De la Rosa MA (2013) New Arabidopsis thaliana cytochrome cpartners: a look into the elusive role of cytochrome cin programmed cell death in plants. Mol Cell Proteomics 12, 3666–3676. 20 Martı´nez-F´ abregas J, Dı´az-Moreno I, Gonz´ alez-Arzola K, Janocha S, Navarro JA, Herv´ as M, Bernhardt R, Vel´ azquez-Campoy A, Dı´az-Quintana A and De la Rosa MA (2014) Structural and functional analysis of novel human cytochrome ctargets in apoptosis. Mol Cell Proteomics 13, 1439–1456. 21 Martı´nez-F´ abregas J, Dı´az-Moreno I, Gonz´ alez-Arzola K, Dı´az-Quintana A and De la Rosa MA (2014) A common signalosome for programmed cell death in humans and plants. Cell Death Dis 5, e1314. 22 Gonz´ alez-Arzola K, Dı´az-Moreno I, Cano-Gonz´ alez A, Dı´az-Quintana A, Vel´ azquez-Campoy A, Moreno- Beltr´ an B, L´ opez-Rivas A and De la Rosa MA (2015) Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβby cytochrome c.Proc Natl Acad Sci USA 112, 9908–9913. 23 Gonz´ alez-Arzola K, Dı´az-Quintana A, Rivero- Rodrı´guez F, Vel´ azquez-Campoy A, De la Rosa MA and Dı´az-Moreno I (2017) Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c.Nucleic Acids Res 45, 2150–2165. 24 Gonz´ alez-Arzola K, Vel´ azquez-Cruz A, Guerra- Castellano A, Casado-Combreras MA, P´ erez-Mejı´as G, Dı´az-Quintana A, Dı´az-Moreno I and De la Rosa MA (2019) New moonlighting functions of mitochondrial cytochrome cin the cytoplasm and nucleus. FEBS Lett 593, 3101–3119. 25 Dı´az-Moreno I, Vel´ azquez-Cruz A, Curran-French S, Dı´az-Quintana A and De la Rosa MA (2018) Nuclear cytochrome c- a mitochondrial visitor regulating damaged chromatin dynamics. FEBS Lett 592, 172–178. 26 Elena-Real CA, Dı´az-Quintana A, Gonz´ alez-Arzola K, Vel´ azquez-Campoy A, Orz´ aez M, L´ opez-Rivas A, Gil- Caballero S, De la Rosa MA and Dı´az-Moreno I (2018) Cytochrome cspeeds up caspase cascade activation by blocking 14-3-3ε-dependent Apaf-1 inhibition. Cell Death Dis 9, 365. 27 Elena-Real CA, Gonz´ alez-Arzola K, P´ erez-Mejı´as G, Dı´az-Quintana A, Vel´ azquez-Campoy A, Desvoyes B, Guti´ errez C, De la Rosa MA and Dı´az-Moreno I (2020) Proposed mechanism for regulation of H 2 O 2 -induced programmed cell death in plants by binding of cytochrome cto 14-3-3 proteins. Plant J 6,74–85. 28 Rivero-Rodrı´guez F, Dı´az-Quintana A, Vel´ azquez-Cruz A, Gonz´ alez-Arzola K, Gavilan MP, Vel´ azquez- Campoy A, Rı´os RM, De la Rosa MA and Dı´az- Moreno I (2021) Inhibition of the PP2A activity by the histone chaperone ANP32B is long-range allosterically regulated by respiratory cytochrome c.Redox Biol 43, 101967. 29 Minakami S, Titani K and Ishikura H (1958) The structure of cytochrome c. II. Properties of acetylated cytochrome c.J Biochem 45, 341–348. 30 Azzi A, Montecucco C and Richter C (1975) The use of acetylated ferricytochrome cfor the detection of superoxide radicals produced in biological membranes. Biochem Biophys Res Commun 65, 597–603. 31 Warburg O (1956) On the origin of cancer cells. Science 123, 309–314. 32 Bazylianska V, Kalpage HA, Wan J, Vaishnav A, Mahapatra G, Turner AA, Chowdhury DD, Kim K, Morse PT, Lee I et al. (2021) Lysine 53 acetylation of cytochrome cin prostate cancer: Warburg metabolism and evasion of apoptosis. Cells 10, 802. 33 Purring-Koch C and McLendon G (2000) Cytochrome cbinding to Apaf-1: the effects of dATP and ionic strength. Proc Natl Acad Sci USA 97, 11928–11931. 34 K¨ onig BW, Osheroff N, Wilms J, Muijsers AO, Dekker HL and Margoliash E (1980) Mapping of the interaction domain for purified cytochrome c 1 on cytochrome c.FEBS Lett 111, 395–398. 35 Kokhan O, Wraight CA and Tajkhorshid E (2010) The binding interface of cytochrome cand cytochrome c 1 in the bc 1 complex: rationalizing the role of key residues. Biophys J 99, 2647–2656. 36 Shimada S, Shinzawa-Itoh K, Baba J, Aoe S, Shimada A, Yamashita E, Kang J, Tateno M, Yoshikawa S and Tsukihara T (2017) Complex structure of cytochrome ccytochrome coxidase reveals a novel protein-protein interaction mode. EMBO J 36, 291–300. 37 P´ erez-Mejı´as G, Olloqui-Sariego JL, Guerra-Castellano A, Dı´az-Quintana A, Calvente JJ, Andreu R, De la Rosa MA and Dı´az-Moreno I (2020) Physical contact between cytochrome c 1 and cytochrome cincreases the driving force for electron transfer. Biochim Biophys Acta 1861, 148277. 38 Olteanu A, Patel CN, Dedmon MM, Kennedy S, Linhoff MW, Minder CM, Potts PR, Deshmukh M and Pielak GJ (2003) Stability and apoptotic activity of 16 FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. recombinant human cytochrome c.Biochem Biophys Res Commun 312, 733–740. 39 Guerra-Castellano A, Dı´az-Moreno I, Vel´ azquez- Campoy A, De la Rosa MA and Dı´az-Quintana A (2016) Structural and functional characterization of phosphomimetic mutants of cytochrome cat threonine 28 and serine 47. Biochim Biophys Acta 1857, 387–395. 40 Guerra-Castellano A, Dı´az-Quintana A, Moreno- Beltr´ an B, L´ opez-Prados J, Nieto PM, Meister W, Staffa J, Teixeira M, Hildebrandt P, De la Rosa MA et al. (2015) Mimicking tyrosine phosphorylation in human cytochrome cby the evolved tRNA synthetase technique. Chem Eur J 21, 15004–15012. 41 Reijenga J, van Hoof A, van Loon A and Teunissen B (2013) Development of methods for the determination of pKa values. Anal Chem Insights 8,53–71. 42 Imai M, Saio T, Kumeta H, Uchida T, Inagaki F and Ishimori K (2016) Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c.Biochem Biophys Res Commun 469, 978– 984. 43 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC and Ferrin TE (2004) UCSF Chimera –a visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612. 44 Papamokos GV, Tziatzos G, Papageorgiou DG, Georgatos SD, Politou AS and Kaxiras E (2012) Structural role of RKS motifs in chromatin interactions: a molecular dynamics study of HP1 bound to a variably modified histone tail. Biophys J 102, 1926– 1933. 45 Autenrieth F, Tajkhorshid E, Baudry J and Luthey- Schulten Z (2004) Classical force field parameters for the heme prosthetic group of cytochrome c.J Comput Chem 25, 1613–1622. 46 Case DA, Belfon K, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TE III, Cruzeiro VWD, Darden TA, Duke RE, Giambasu G et al. (2020) AMBER 2020. University of California, San Francisco, CA. 47 Eastman P, Swails J, Chodera JD, McGibbon RT, Zhao Y, Beauchamp KA, Wang LP, Simmonett AC, Harrigan MP, Stern CD et al. (2017) OpenMM 7: Rapid development of high performance algorithms for molecular dynamics. PLoS Comput Biol 13, e1005659. 48 Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE and Simmerling C (2015) ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput 11, 3696–3713. 49 Izadi S, Anandakrishnan R and Onufriev AV (2014) Building water models: A different approach. J Phys Chem Lett 5, 3863–3871. 50 Roe DR and Cheatham TE (2013) PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput 9, 3084–3095. 51 Lee W, Tonelli M and Markley JL (2015) NMRFAMSPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327. 52 M¨ uller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M and Randau L (2020) Bio-Layer Interferometry analysis of the target binding activity of CRISPR-Cas effector complexes. Front Mol Biosci 7, 98. 53 Frezza C, Cipolat S and Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2, 287–295. 54 Guerra-Castellano A, Dı´az-Quintana A, P´ erez-Mejı´as G, Elena-Real CA, Gonz´ alez-Arzola K, Garcı´a-Mauri˜ no SM, De la Rosa MA and Dı´az-Moreno I (2018) Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria. Proc Natl Acad Sci USA 115, 7955–7960. 55 Isom AL, Barnes S, Wilson L, Kirk M, Coward L and Darley-Usmar V (2004) Modification of cytochrome c by 4-hydroxy- 2-nonenal: Evidence for histidine, lysine, and arginine-aldehyde adducts. J Am Soc Mass Spectrom 15, 1136–1147. 56 Santucci R and Ascoli F (1997) The Soret circular dichroism spectrum as a probe for the heme Fe(III)- Met(80) axial bond in horse cytochrome c.J Inorg Biochem 68, 211–214. 57 Banci L, Bertini I, Gray HB, Luchinat C, Reddig T, Rosato A and Turano P (1997) Solution structure of oxidized horse heart cytochrome c.Biochemistry 36, 9867–9877. 58 Blauer G, Sreerama N and Woody RW (1993) Optical activity of hemoproteins in the Soret region. Circular dichroism of the heme undecapeptide of cytochrome c in aqueous solution. Biochemistry 32, 6674–6679. 59 Boffi F, Bonincontro A, Cinelli S, Congiu Castellano A, De Francesco A, Della Longa S, Girasole M and Onori G (2001) pH-dependent local structure of ferricytochrome cstudied by x-ray absorption spectroscopy. Biophys J 80, 1473–1479. 60 Ying T, Zhong F, Xie J, Feng Y, Wang ZH, Huang ZX and Tan X (2009) Evolutionary alkaline transition in human cytochrome c.J Bioenerg Biomembr 41, 251– 257. 61 Wilson MT and Greenwood C (1996) The alkaline transition in ferricytochrome c.InCytochrome c: A Multidisciplinary Approach (Scott RA and Mauk AG, eds), pp. 611–634. University Science Books, Sausalito, CA. 62 Assfalg M, Bertini I, Dolfi A, Turano P, Mauk AG, Rosell FI and Gray HB (2003) Structural model for an alkaline form of ferricytochrome c.J Am Chem Soc 125, 2913–2922. 17FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c 63 Schejter A and George P (1964) The 695-mμband of ferricytochrome cand its relationship to protein conformation. Biochemistry 3, 1045–1049. 64 Dickerson R and Timkovich R (1975) Cytochrome c.In The Enzymes (Boyer P, ed), pp. 397–472. Academic Press, New York, NY. 65 Garcı´a-Heredia JM, Dı´az-Quintana A, Salzano M, Orz´ aez M, P´ erez-Pay´ a E, Teixeira M, De la Rosa MA and Dı´az-Moreno I (2011) Tyrosine phosphorylation turns alkaline transition into a biologically relevant process and makes human cytochrome cbehave as an anti-apoptotic switch. J Biol Inorg Chem 16, 1155–1168. 66 Poulos TL (1996) The role of the proximal ligand in heme enzymes. J Biol Inorg Chem 1, 356–359. 67 Maity H, Maity M and Englander SW (2004) How cytochrome cfolds, and why: submolecular foldon units and their stepwise sequential stabilization. J Mol Biol 343, 223–233. 68 Maity H, Rumbley JN and Englander SW (2006) Functional role of a protein foldon –an Omega-loop foldon controls the alkaline transition in ferricytochrome c.Proteins 63, 349–355. 69 Dragomir I, Hagarman A, Wallace C and Schweitzer- Stenner R (2007) Optical band splitting and electronic perturbations of the heme chromophore in cytochrome cat room temperature probed by visible electronic circular dichroism spectroscopy. Biophys J 92, 989–998. 70 Fisher WR, Taniuchi H and Anfinsen CB (1973) On the role of heme in the formation of the structure of cytochrome c.J Biol Chem 248, 3188–3195. 71 Kirby EP and Steiner RF (1970) The tryptophan microenvironments in apomyoglobin. J Biol Chem 245, 6300–6306. 72 Jameson C (1996) Understanding NMR chemical shifts. Annu Rev Phys Chem 47, 135–169. 73 W¨ uthrich K (1986) NMR of proteins and nucleic acids. John Wiley and Sons, Inc, New York, NY. 74 Rieder R and Bosshard HR (1980) Comparison of the binding sites on cytochrome cfor cytochrome c oxidase, cytochrome bc 1 , and cytochrome c 1 . Differential acetylation of lysyl residues in free and complexed cytochrome c.J Biol Chem 255, 4732–4739. 75 Yu H, Lee I, Salomon AR, Yu K and H¨ uttemann M (2008) Mammalian liver cytochrome cis tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration. Biochim Biophys Acta 1777, 1066–1071. 76 Pecina P, Borisenko GG, Belikova NA, Tyurina YY, Pecinova A, Lee I, Samhan-Arias AK, Przyklenk K, Kagan VE and H¨ uttemann M (2010) Phosphomimetic substitution of cytochrome ctyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation. Biochemistry 49, 6705–6714. 77 Hildick-Smith GJ, Downey MC, Gretebeck LM, Gersten RA and Sandwick RK (2011) Ribose 5- Phosphate glycation reduces cytochrome crespiratory activity and membrane affinity. Biochemistry 50, 11047– 11057. 78 Aluri HS, Simpson DC, Allegood JC, Hu Y, Szczepanek K, Gronert S, Chen Q and Lesnefsky EJ (2014) Electron flow into cytochrome ccoupled with reactive oxygen species from the electron transport chain converts cytochrome cto a cardiolipin peroxidase: Role during ischemia-reperfusion. Biochim Biophys Acta 1840, 3199–3207. 79 Mahapatra G, Varughese A, Ji Q, Lee I, Liu J, Vaishnav A, Sinkler C, Kapralov AA, Moraes CT, Sanderson TH et al. (2017) Phosphorylation of cytochrome cthreonine 28 regulates electron transport chain activity in kidney: Implications for amp kinase. J Biol Chem 292,64–79. 80 Moreno-Beltr´ an B, Guerra-Castellano A, Dı´az- Quintana A, Del Conte R, Garcı´a-Mauri˜ no SM, Dı´az- Moreno S, Gonz´ alez-Arzola K, Santos-Oca˜ na C, Vel´ azquez-Campoy A, De La Rosa MA et al. (2017) Structural basis of mitochondrial dysfunction in response to cytochrome cphosphorylation at tyrosine 48. Proc Natl Acad Sci USA 114, E3041–E3050. 81 Smith HT, Ahmed AJ and Millett F (1981) Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase. J Biol Chem 256, 4984–4990. 82 Lange C and 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. 83 Hunte C, Solmaz S and Lange C (2002) Electron transfer between yeast cytochrome bc 1 complex and cytochrome c: a structural analysis. Biochim Biophys Acta - Bioenerg 1555,21–28. 84 Sakamoto K, Kamiya M, Imai M, Shinzawa-Itoh K, Uchida T, Kawano K, Yoshikawa S and Ishimori K (2011) NMR basis for interprotein electron transfer gating between cytochrome cand cytochrome coxidase. Proc Natl Acad Sci USA 108, 12271–12276. 85 Rodrı´guez-Rold´ an V, Garcı´a-Heredia JM, Navarro JA, Herv´ as M, De la Cerda B, Molina-Heredia FP and De la Rosa MA (2006) A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome ctowards cytochrome coxidase. Biochem Biophys Res Commun 346, 1108–1113. 86 Olloqui-Sariego JL, Zakharova G, Poloznikov AA, Calvente JJ, Hushpulian DM, Gorton L and Andreu R (2019) The Fe (III)/Fe(II) redox couple as a probe of immobilized tobacco peroxidase: effect of the immobilization protocol. Electrochim Acta 299,55–61. 87 Sato W, Hitaoka S, Inoue K, Imai M, Saio T, Uchida T, Shinzawa-Itoh K, Yoshikawa S, Yoshizawa K and Ishimori K (2016) Energetic mechanism of cytochrome c-cytochrome c oxidase electron transfer complex formation under turnover conditions revealed by 18 FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al. mutational effects and docking simulation. J Biol Chem 291, 15320–21533. 88 Medina M, Herv´ as M, Navarro JA, De la Rosa MA, G´ omez-Moreno C and Tollin G (1992) A laser flash absorption spectroscopy study of Anabaena sp. PCC 7119 flavodoxin photoreduction by photosystem I particles from spinach. FEBS Lett 313, 239–242. 89 De la Cerda B, Navarro JA, Herv´ as M and De la Rosa MA (1997) Changes in the reaction mechanism of electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 as induced by site-directed mutagenesis of the copper protein. Biochemistry 36, 10125–10130. 90 Sun J, Xu W, Herv´ as M, Navarro JA, Rosa MA and Chitnis PR (1999) Oxidizing side of the cyanobacterial photosystem I. Evidence for interaction between the electron donor proteins and a luminal surface helix of the PsaB subunit. J Biol Chem 274, 19048–19054. 91 Fraz˜ ao C, Enguita FJ, Coelho R, Sheldrick GM, Navarro JA, Herv´ as M, De la Rosa MA and Carrondo MA (2001) Crystal structure of low-potential cytochrome c 549 from Synechocystis sp. PCC 6803 at 1.21 ˚ A resolution. J Biol Inorg Chem 6, 324–332. 92 Dı´az-Moreno I, Dı´az-Quintana A, De la Rosa MA, Crowley PB and Ubbink M (2005) Different modes of interaction in cyanobacterial complexes of plastocyanin and cytochrome f.Biochemistry 44, 3176–3183. 93 Dı´az-Moreno I, Dı´az-Quintana A, Molina-Heredia FP, Nieto PM, Hansson O, De la Rosa MA and Karlsson BG (2005) NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c 6 .J Biol Chem 280, 7925–7931. 94 Dı´az-Moreno I, Dı´az-Quintana A, Ubbink M and De la Rosa MA (2005) An NMR-based docking model for the physiological transient complex between cytochrome fand cytochrome c 6 .FEBS Lett 579, 2891–2896. 95 Go˜ ni G, Herguedas B, Herv´ as M, Peregrina JR, De la Rosa MA, G´ omez-Moreno C, Navarro JA, Hermoso JA, Martı´nez-J´ ulvez M and Medina M (2009) Flavodoxin: a compromise between efficiency and versatility in the electron transfer from Photosystem I to Ferredoxin-NADP(+) reductase. Biochim Biophys Acta - Bioenerg 1787, 144–154. Supporting information Additional Supporting Information may be found online in the Supporting Information section at the end of the article. Fig. S1. Expression and purification of WT and mutant cytochrome cspecies. (A) Left panel: SDSPAGE of the 15 N-labelled WT, K8A, K8Q, K53A and K53Q Ccsamples used for NMR experiments (5 μgof protein loaded in each lane). The bands within the red rectangle (below 15 kDa) correspond to the different Ccspecies. M: Molecular mass markers. Right panel: Western blot of purified WT, K8A, K8Q, K53A and K53Q Ccshowing the detection of the Ccband in each lane. (B) Tryptic digestion of proteins extracted from the bands of an SDS-PAGE similar to that in (A) but loaded with 14 N-labelled Ccsamples. The calculated masses of AIFIMK, QIFIMK, KTGQAPGYSYTAANANK and KTGQAPGYSYTAANQNK fragments are 722, 779, 1741 and 1799 Da, respectively. Fig. S2. Secondary structure analysis of WT and mutant cytochrome cspecies. Percentage of secondary structure for oxidized WT, K8A, K8Q, K53A and K53Q Ccis calculated from far-UV CD data with the CDPro software package (SP43, SMP50 and CLSTR reference sets) [1]. The results are expressed as the mean SD. Fig. S3. Alkaline transition of WT and mutant cytochrome cspecies. Electronic absorption spectra were recorded at different pH values (left panels), and titration curves were determined by following the absorbance changes at 695 nm (right panels) of the oxidized species. Process reversibility was checked by recording the last spectrum at the initial pH value. The full data set was fitted to the Henderson–Hasselbalch equation to calculate the pK a values (red lines; see details in Methods section). Fig. S4. Molecular dynamic simulations of WT and mutant cytochrome cspecies. Average of the Met80Fe distance (upper) and solvent accessibility of the heme group (lower) within the last 50 ns of their respective MD trajectories are shown for WT, K8A, K8Q, K8AcK, K53A, K53Q and K53AcK Ccspecies. The results are expressed as the mean SD. Fig. S5. Thermal stability of WT and mutant cytochrome cspecies. The first principal component (P1) of CD and fluorescence spectra of oxidized WT, K8A, K8Q, K53A and K53Q Ccrecorded at varying temperature is plotted. Fig. S6. Superimposition of 2D 1 H– 15 N HSQC NMR spectra of WT (black) and K8A (red) cytochrome c. Insets: Zooms of the signals framed in yellow boxes. The green arrows stand for the CSPs of mutant Ccas compared with the WT species. The yellow circle denotes the mutated residue. Fig. S7. Superimposition of 2D 1 H– 15 N HSQC NMR spectra of WT (black) and K8Q (blue) cytochrome. Insets: Zooms of the signals framed in yellow boxes. The red arrows stand for the CSPs of mutant Ccas compared with the WT species. The red dotted line shows the 15 N chemical shift at which the 1 H of the 19FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. I. M´ arquez et al.Acetyl-mimetic mutants of cytochrome c Q8 ε-amine group appears. The yellow circle denotes the mutated residue. Fig. S8. Superimposition of 2D 1 H– 15 N HSQC NMR spectra of WT (black) and K53A (green) cytochrome c.Insets: Zooms of the signals framed in yellow boxes. The red arrows stands for the CSPs of mutant Ccas compared with the WT species. The yellow circle surrounds the mutated residue. Fig. S9. Superimposition of 2D 1 H– 15 N HSQC NMR spectra of WT (black) and K53Q (purple) cytochrome c.Insets: Zooms of the signals framed in yellow boxes. The green arrows stand for the CSPs of mutant Ccas compared with the WT species. The green dotted line shows the 15 N chemical shift at which the 1 H of the Q53 ε-amine group appears. The yellow circle surrounds the mutated residue. Fig. S10. Interaction of WT and mutant cytochrome c with cytochrome c 1 as determined by BLI. Wavelength shift over time (left panels) and calculated dissociation equilibrium constant (K D ;right panels) for the interaction between reduced Cc 1 and reduced WT, K8Q or K53Q Ccwere determined at increasing Ccconcentration. The dotted gray line in the left panels indicates the time at which the wavelength shifts were taken for calculation of the K D values in the right panels. The data set was fitted to a non-linear equation to obtain the K D values (red lines; see details in Methods section). The results are expressed as the mean SD. Fig. S11. Electrochemical characterization of WT and mutant cytochrome c. (A) Cyclic voltammograms of reduced Ccspecies immobilized onto a chemically modified polycrystalline gold electrode (scan rate: 0.1 V s -1 ). (B) Midpoint redox potential and (C) electron transfer rate constant for immobilized Ccas determined at varying temperature under the same experimental conditions as in panel A. The results are expressed as the mean SD. Fig. S12. Binding sites for cytochrome con complex IV at the respiratory supercomplex. The representative structure of WT Ccat the distal site of complex IV is in orange, where Lys53 (green) points towards the complex IV surface (light gray), and that at the proximal site is in red, where Lys8 (blue) points towards the complex IV surface. Table S1. Primers used in the design of cytochrome c mutants. Table S2. BLI protocol for cytochrome c 1 –cytochrome cinteraction. 20 FEBS Open Bio (2021) ª2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. Acetyl-mimetic mutants of cytochrome cI. M´ arquez et al.