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Title:Biochemical characterization of respiratory chain flavoproteins
Author(s):Lencina, Andrea Mariana
Director of Research:Gennis, Robert B.
Doctoral Committee Chair(s):Gennis, Robert B.
Doctoral Committee Member(s):Imlay, James A.; Chen, Lin-Feng; Oldfield, Eric
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
electron transport chain
nadh dehydrogenase
sulfide quinone reductase
coenzyme A disulfide reductase
Streptococcus agalactiae
Caldivirga maquilingensis
Thermus thermophilus
Abstract:Respiring organisms derive energy mainly from electron transfer reactions that are coupled to the translocation of ions across the cell membrane. This process is performed by a group of membrane-localized protein complexes that hold different cofactors with redox properties, such as flavins, hemes, and other iron-containing centers. These redox complexes constitute the electron transport or respiratory chain. This thesis will cover the study of three different flavin-containing proteins related to the electron transport chain: • The type 2 NADH dehydrogenase (NDH-2) from Streptococcus agalactiae • The sulfide:quinone oxidoreductase (SQR) from the archaeon Caldivirga maquilingensis • The NDH-2 from Thermus thermophilus, now re-classified as a coenzyme A disulfide reductase (CoADR) NDH-2 catalyzes the same reaction (NADH:quinone oxidoreductase) as complex I, however it is unable to translocate protons across the membrane. Absent in mammalian mitochondria and the main or only respiratory NADH dehydrogenase present in certain pathogens, it has recently gained interest as a putative drug target. S. agalactiae is the major cause of meningitis and sepsis in a newborn’s first week of life in the United States. This organism encodes an NDH-2 as its only NADH dehydrogenase, and respiration has been shown to be important for virulence in an animal model. Here, NDH-2 was found to be the sole point of entry for electrons to the S. agalactiae respiratory chain. Additionally, in a mouse model of infection, mutant strains lacking NDH-2 showed significant attenuation for organ colonization. The protein was expressed, purified, biochemically characterized and used for in vitro screening of inhibitors. Several compounds were found to block activity of the protein and one even inhibited respiration in whole cells. SQRs (H2S + Q → S0 + QH2) are ubiquitous enzymes that can provide electrons to respiratory or photosynthetic electron transfer chains, besides having roles in sulfide detoxification and homeostasis. C. maquilingensis is a hyperthermophilic archaeon encoding a type III SQR, which lacks a cysteine residue involved in flavin binding in other SQR classes. This protein was expressed, purified and biochemically characterized, showing sulfide oxidation in the presence of quinone. The mode of membrane binding of CmSQR was studied by constructing truncated versions of the enzyme, lacking one or both amphipathic C-terminal helices. These variants were expressed as inactive soluble enzymes and lack of activity was attributed to the mutation of a single leucine residue, presumably involved in quinone interaction. Further mutations were made based on a homology model for CmSQR and crystal structures available for other SQR types, to characterize the quinone and flavin binding sites. Lastly, the oligomeric state of CmSQR was studied and the protein determined to be a dimer by different methods, including gel filtration, native gel and crosslinking. T. thermophilus aerobic NDH-2 exhibits very low activity (1 electron/sec) for NADH oxidation in the presence of quinone analogs. Recently, the crystal structure for TtNDH-2 was solved indicating strong similarity to Pyrococcus horikoshii CoADR (NADH: Coenzyme A disulfide oxidoreductase) and making us re-evaluate this assignment. Biochemical assays showed the enzyme CoADR activity is 6-fold greater than its NDH-2 activity. Kinetic parameters are also similar to other known CoADRs, and the enzyme can be inhibitied by a specific active-site cysteine modifier.
Issue Date:2018-07-10
Rights Information:Copyright 2018 Andrea Lencina
Date Available in IDEALS:2018-09-27
Date Deposited:2018-08

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