Biochemical characterization of C-family heme copper oxygen reductase

Ahn, Young O

Permalink

https://hdl.handle.net/2142/78726 Copy

Description

Title

Biochemical characterization of C-family heme copper oxygen reductase

Author(s)

Ahn, Young O

Issue Date

2015-04-17

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

Gennis, Robert B.

Doctoral Committee Chair(s)

Gennis, Robert B.

Committee Member(s)

• Lu, Yi
• Nair, Satish K.
• Schuler, Mary A.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• cbb3
• heme copper oxygen reductase
• bioenergetics
• vibrio cholerae

Abstract

Heme copper oxygen reductases (HCOs) are key enzymes for aerobic respiration. As a terminal electron acceptor, HCO catalyzes the sequential transfer of four electrons from reduced cytochromes c on the P-side of the membrane, and four protons from the cytosol on the N-side, to molecular O2 bound at the active site that results in the production of water. The excess energy released in this reaction is used to pump protons from the N-side to the P-side. These processes establish an electrochemical proton gradient across the membrane that is utilized by ATP synthase for ATP production. The HCO superfamily is primarily classified into A-, B- and C-families. The C-family HCOs, which are found exclusively in prokaryotes including a number of pathogenic bacteria, are evolutionally quite distant from the A-family HCOs present in human mitochondria. There are distinct differences between the A- and C-family that make the C-family HCOs in pathogens excellent drug targets. However, much less is known about the molecular mechanism of the C-family HCOs. This thesis is focused on the biochemical characterization of C-family HCO from Vibrio cholerae. Structure analysis, sequence analysis, molecular dynamics (MD) simulations and site-directed mutagenesis revealed that the C-family HCOs utilize only a single proton channel (KC-channel), in contrast to the A-family HCOs that use two proton channels. Our research also confirmed that the KC-channel begins at the cytoplasm facing E49 in the CcoP subunit, and ends at Y321 near the active site in the CcoN subunit, and includes internal water molecules and several conserved polar residues (e.g. Y227, S244 and N293) that form a hydrogen bonded network to facilitate proton diffusion. Further characterizations using UV-visible and Resonance Raman spectroscopies, and CO recombination kinetics indicated that the active site was conformationally linked to changes of residues within the KC-channel. The detailed reaction mechanism during oxidation was further examined using the flow-flash technique. The results suggested that the proton transfer process involves two-steps: 1) proton transfer from an internal proton donor to the active site and 2) proton transfer from the bulk solution via the entrance (E49) of the KC-channel for rapid reprotonation of the internal proton donor. Y321, located further up the KC-channel, was suggested to be a bifurcation point, where protons are diverged to either the active site to reduce oxygen to water or the proton-loading site (PLS) for pumping to the P-side. This is supported by our observations that Y321 contributes to the fast unidirectional proton transfer in the KC-channel, thereby preventing protons from diffusing back to the cytosol. We also observed that the proposed proton exit pathway from Y321 to a putative PLS (N337/H341) for proton pumping is connected to the conformation of both the active site and the CcoP subunit implying its crucial role in the reaction. Additionally, the function of the CcoP subunit (a membrane bound diheme cytochrome c), which contributes to the distinct structural properties of the C-family HCOs, was examined. We propose that the CcoP subunit functions to extend both electron and proton wires by providing entrances for each pathway, and that the transmembrane helices of CcoP are required for the assembly/stability of the enzyme. It is concluded that in the C-family HCOs the unique structural features of electron and proton transfer pathways are correlated with the specific enzyme functions and mechanisms. Thus our study provides a fundamental insight into the mechanisms of C-family HCOs and may contribute to the development of new drugs targeting C-family HCOs in pathogens.

Graduation Semester

2015-5

Type of Resource

text

Permalink

http://hdl.handle.net/2142/78726

Copyright and License Information

Copyright 2015 Young Ahn

Owning Collections