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

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 acetylationinduced 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. Nuclear magnetic resonance 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.

The role of lysine acetylation in the function of mitochondrial ribosomal protein L12

“…Arginine is similar in size and charge to lysine, but is unable to be acetylated. It is important to note that these protein mutants do not perfectly represent the acetylated or deacetylated forms of MRPL12; however, acetyl mimics have been routinely used to characterize the impact of acetylation on protein function 33,34 …”

“…It is important to note that these protein mutants do not perfectly represent the acetylated or deacetylated forms of MRPL12; however, acetyl mimics have been routinely used to characterize the impact of acetylation on protein function. 33,34 Acetyl and deacetyl MRPL12 mimics were recombinantly expressed and purified. Purified proteins were used in fluorescence polarization experiments to determine whether acetylation of MRPL12 altered the ability of POLRMT to bind the light strand promoter region.…”

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochromecin cells

“…2 C , the cyt c -cardiolipin–containing liposomes inhibit the binding of mAb 1D3 and R1D3 to the adsorbed cyt c on the ELISA plate, whereas no inhibition was observed for the cyt c /DOPC liposome and for the liposomes alone. Finally, several cyt c mutants that mimic PTMs (phosphorylation and acetylation) at different residues were tested ( 3 , 8 , 10 – 12 , 15 ). As shown in Fig.…”

“…Phosphorylation of cyt c at multiple Ser, Thr, and Tyr sites has been previously shown to occur in different tissues as well as acetylation. These PTMs are involved in the regulation of cell respiration and other mitochondrial processes ( 3 , 4 , 8 – 15 ). Using cyt c mutants that mimic different phosphorylation and acetylation sites, it was shown that the mAb 1D3/R1D3 fails to recognize them in agreement with the spectroscopic studies that indicate the presence of the M80–Fe bond in all these cyt c mimetics ( Fig.…”

“…The translocation of cyt c to the cytoplasm is provoked by different death stimuli and leads to the assembly of the apoptosome, with the final activation of caspase 3 resulting in the execution of intrinsic apoptosis ( 2 ). The activity of cyt c can be modulated by reversible posttranslational modifications (PTMs), such as phosphorylation at serine, threonine, and tyrosine residues or lysine acetylation, which impact different biochemical activities, such as the inhibition of caspase 3 and cyt c oxidase activities ( 3 – 15 ).…”