Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Abstract

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…This mutation mimics protein phosphorylation at Tyr48 by adding a negative charge and slightly increasing the side-chain size while keeping the aromatic ring. A recent spectroscopic analysis of Y48pCMF Cc showed a singular shift of the typical alkaline transition pK a to physiological pH values (31), as is the case with the Y48E Cc mutant (26,27). Here, we report NMR-based structure computations that indicate that Tyr48 phosphorylation maintains the core foldon of Cc but increases internal motions in the loops Ω NY , Ω R , and Ω G .…”

“…It is well-known that the catalytic step can be tuned by conformational changes of Cc upon binding to CcO (52). Strikingly, these changes resemble the prominent internal dynamics of Y48pCMF Cc, which could also explain not only the change in E 0 but also the shift in the alkaline transition pK a of oxidized Y48pCMF Cc (31). Therefore, the transition from reduced to oxidized Y48pCMF Cc, which is accompanied by a change in the iron axial ligand at physiological pH, is expected to have a high-energy barrier (82).…”

“…Thus, these posttranslational modifications alter both mitochondrial and cytoplasmic functions of Cc. Tyr48 phosphorylation also affects the physical-chemical properties of Cc, such as its alkaline transition and midpoint reduction potential (E 0 ) (27,31). Understanding the origin of these effects and how they affect Cc activities requires solving the 3D conformation of the phosphorylated species.…”

“…The only exception was the K D value for the oxidized Cc 1 -Y48pCMF Cc complex, at the proximal site of Cc 1 , which was approximately four times lower than that for the oxidized Cc 1 -WT Cc adduct at pH 8.5. ITC measurements of the oxidized Cc 1 -Y48pCMF Cc complex were not run at pH 7.4 because the pK a for the alkaline transition, which is specific to the ferric state (53), shifts to neutral pH upon Tyr48 phosphorylation (31). Hence, the WT and phosphomimetic forms of oxidized Cc can differ in their sixth axial ligand at pH 7.4 (SI Appendix, Table S5).…”

Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c

“…C c is an electron carrier in the mitochondrial electron transport chain; and its function is tightly regulated by post-translational modifications (36–38). Interestingly, SET/TAF-Iβ, the protein analogous to NRP1 in humans, is also targeted by C c in human cell nuclei following DNA damage (39,40).…”