Abstract
Post‐translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and 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 c with 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 c to 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 c oxidase.
Highlights
A principal mitochondrial protein affected by Post-translational modifications (PTMs) is respiratory cytochrome c (Cc), which has been described as a target for lysine acetylation [14]
Physicochemical features of cytochrome c are mainly unchanged in the two acetyl-mimetic mutants
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
Summary
Abbreviations A, pre-exponential factor; AcK, acetyl-lysine; ANOVA, one-way analysis of variance; BLI, Bio-layer interferometry; Cc, cytochrome c; Cc1, cytochrome c1; CcO, cytochrome c oxidase; CIII, complex III; CIV, complex IV; CSP, chemical shift perturbation; DMEM, Dulbecco’s modified Eagle’s medium; E. coli, Escherichia coli; E0, midpoint reduction potential; E1/2, midpoint redox potential value; ETC, electron transport chain; HSQC, heteronuclear single quantum correlation; ITC, isothermal titration calorimetry; KD, dissociation equilibrium constant; ks, 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; Tm, midpoint melting temperature; TOCSY, total correlation spectroscopy; UV, ultraviolet; Vis, visible; WT, wild-type; ε, extinction coefficient. Post-translational modifications (PTMs) have traditionally been associated with mechanisms that cells use to regulate and expand the functionality of their biomolecules [1,2,3] 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. 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 [8,9,10,11]. Several internal and external cell stimuli can induce the translocation of Cc to other cell compartments (nucleus, vacuoles, zymogen granules, cytosol, and even the rough endoplasmic reticulum), where Cc behaves as a moonlighting protein able to interact with an ample set of non-redox proteins [18,19,20,21,22,23,24,25,26,27,28]
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