Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.
Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.
Yu
Hong
H
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 3214 Scott Hall, 540 E. Canfield, Detroit, MI 48201, USA.
Lee
Icksoo
I
Salomon
Arthur R
AR
Yu
Kebing
K
Hüttemann
Maik
M
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D045304:Cytochromes c / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D010101:Oxygen Consumption / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D019000:Phosphotyrosine; D014443:Tyrosine; D045304:Cytochromes c; D003576:Electron Transport Complex IV