TY - JOUR
T1 - Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components
AU - Ambrus, Attila
AU - Nemeria, Natalia S.
AU - Torocsik, Beata
AU - Tretter, Laszlo
AU - Nilsson, Mattias
AU - Jordan, Frank
AU - Adam-Vizi, Vera
N1 - Funding Information:
We are grateful to Drs. Hetalben Patel, Sowmini Kumaran, Junjie Wang, all from Rutgers, and Da Jeong Shim and Edgardo T. Farinas (New Jersey Institute of Technology) for providing purified proteins to this project, and Mulchand S. Patel and his group for providing cells harboring plasmids of the hPDHc components. Financial support is gratefully acknowledged from the Hungarian Academy of Sciences (MTA grant 02001 to A-V.V.), the Hungarian Scientific Research Fund (OTKA, grant 112230 to A-V. V.), the Hungarian Brain Research Program (grant KTIA_13_NAP-A-III/6. to A-V.V.), the Bolyai and the Fulbright Fellowships (to A.A.), and the NIH (NIH-GM-050380 and NIH-GM-116077 to F.J.).
Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess 'moonlighting' activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca2+, ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.
AB - Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess 'moonlighting' activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca2+, ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.
KW - 2-oxoglutarate dehydrogenase complex
KW - Alpha-ketoglutarate dehydrogenase complex
KW - E3 deficiency
KW - Oxidative stress
KW - Pyruvate dehydrogenase complex
KW - Reactive oxygen species
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U2 - 10.1016/j.freeradbiomed.2015.10.001
DO - 10.1016/j.freeradbiomed.2015.10.001
M3 - Article
C2 - 26456061
AN - SCOPUS:84944391651
SN - 0891-5849
VL - 89
SP - 642
EP - 650
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
ER -