title: Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components.
abstract: 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 Ca(2+), 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.
authors.affiliation: Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Ambrus
authors.affiliation: Department of Chemistry, Rutgers, the State University, Newark, NJ 07102, USA.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Nemeria
authors.affiliation: Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary.
authors.forename: Beata
authors.initials: B
authors.lastname: Torocsik
authors.affiliation: Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Tretter
authors.affiliation: Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary.
authors.forename: Mattias
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: Department of Chemistry, Rutgers, the State University, Newark, NJ 07102, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
authors.affiliation: Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary. Electronic address: adam.veronika@med.semmelweis-univ.hu.
authors.forename: Vera
authors.initials: V
authors.lastname: Adam-Vizi
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D007655:Ketoglutarate Dehydrogenase Complex
keywords: 2-oxoglutarate dehydrogenase complex; Alpha-ketoglutarate dehydrogenase complex; E3 deficiency; Oxidative stress; Pyruvate dehydrogenase complex; Reactive oxygen species