Characterization of alternative enzymes in bacterial respiration

Venkatakrishnan, Padmaja

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https://hdl.handle.net/2142/90759 Copy

Description

Title

Characterization of alternative enzymes in bacterial respiration

Author(s)

Venkatakrishnan, Padmaja

Issue Date

2016-04-18

Director of Research (if dissertation) or Advisor (if thesis)

Gennis, Robert B.

Doctoral Committee Chair(s)

Gennis, Robert B.

Committee Member(s)

• Crofts, Antony R.
• Imlay, James A.
• Lu, Yi

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• respiration
• Nicotinamide adenine dinucleotide (NADH)
• proton motive force
• supercomplex

Abstract

"All organisms require energy for critical life processes, starting from the unicellular prokaryotes to the multicellular and highly complex eukaryotes and they largely derive this as a byproduct of respiration. Bacteria are known for their branched and highly modular aerobic and anaerobic respiratory chains. The branching refers to the presence of multiple respiratory enzymes that provide electrons to the electron transport chain as well as multiple enzymes that provide electrons to different terminal electron acceptors. The type II NADH dehydrogenases (NDH-2s) represent one such alternative enzyme. They are membrane-associated enzymes that oxidize NADH and reduce quinone, similar to the canonical Complex I in mitochondria. However unlike the Complex I they do not contribute to energy conservation. The first part of this thesis focuses on the two types of NDH-2s in Thermus thermophilus that are expressed under different growth conditions. One NDH-2 had been isolated previously from the obligate aerobic strain of T. thermophilus, HB27. The gene considered most likely to encode this enzyme was identified and cloned, and the enzyme was isolated and characterized. A different NDH-2 was isolated from the NAR1 strain of T. thermophilus that can be grown anaerobically and catalyze denitrification. Interestingly, in this strain the putative NDH-2 encoded by a gene (nrcN) that is part of an operon (nrcDEFN). The operon is proposed to encode for a protein complex specifically required for nitrate reduction. The NrcN was shown to function independently of the other components of the putative Nrc complex. The biochemical properties of the two NDH-2 enzymes were compared. Another level of variation within electron transport chains is at the level of the quinone: cytochrome c oxidoreductase. In many prokaryotes and in all eukaryotes, this reaction is catalyzed by the bc1 complex, also known as Complex III. An alternative enzyme called ""alternative complex III"" or ACIII is also found to be widely distributed in the bacterial kingdom. This complex has only been isolated from two thermophilic bacteria so far. The second part of this thesis focuses on the ACIII from Flavobacterium johnsoniae. The genome of this organism encodes two cytochrome c oxidases but does not have the genes for the bc1 complex. The essential reaction normally carried out by the bc1 complex is, instead, catalyzed by the ACIII. It’s not known if the ACIII is capable coupling its reaction to the generation of a proton motive force. To qualitatively test for the generation of a proton motive force by the ACIII, lipophilic fluorescent dyes that respond to changes in either the pH gradient or the potential gradient across the membranes were used with spheroplasts and membrane vesicles of F. johnsoniae. Our preliminary experiments with both the membrane vesicles and spheroplasts show no evidence of contribution to the proton motive force by the ACIII. An attempt was made to isolate the ACIII from the membranes of Flavobacterium johnsoniae, but it was instead found to co-purify with the aa3 oxidase as a stable supercomplex. This supercomplex was characterized and its components were identified. The supercomplex was initially isolated in the detergent, dodecyl maltoside (DDM), but did not have a homogenous population. Therefore, efforts were made to isolate the supercomplex in a detergent - free manner using the styrene maleic acid (SMA) copolymer. The preparation of the supercomplex in the SMA had a more homogenous population, which would be suitable for use in structural studies."

Graduation Semester

2016-05

Type of Resource

text

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http://hdl.handle.net/2142/90759

Copyright and License Information

Copyright 2016 Padmaja Venkatakrishnan

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