The Binding Pockets of QA and QB in the Photosynthetic Reaction Center of Rba. sphaeroides Probed by Pulsed EPR

Martin, Erik W.

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https://hdl.handle.net/2142/24256 Copy

Description

Title

The Binding Pockets of QA and QB in the Photosynthetic Reaction Center of Rba. sphaeroides Probed by Pulsed EPR

Author(s)

Martin, Erik W.

Issue Date

2011-05-25T14:59:33Z

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

Wraight, Colin A.

Doctoral Committee Chair(s)

Wraight, Colin A.

Committee Member(s)

• Gennis, Robert B.
• Crofts, Antony R.
• Belford, R. Linn

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Computnl Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Reaction Center
• Electron paramagnetic resonance (EPR)
• Electron spin echo envelope modulation (ESEEM)
• HYSCORE
• Quinone
• Rhodobacter sphaeroides
• redox
• Own n-layered integrated molecular orbital and molecular mechanics (ONIOM)
• Molecular Dynamics (MD)

Abstract

The bacteria Rhodobacter sphaeroides uses photosynthetic reaction centers to convert light into chemical energy. Light absorption by the reaction center results in electron transfer through a series of cofactors, the final two are identical ubiquinones (QA and QB). Both ubiquinones can be stabilized in their reduced (semiquinone) state allowing for their binding pockets to be studied with EPR (electron paramagnetic resonance) spectroscopy. Using the pulsed EPR technique, ESEEM (electron spin echo envelope modulation) the interactions between the reaction center semiquinones and their environment were studied for wild type and a mutant reaction centers. Nitrogen ESEEM modulations showed different nuclear couplings between the QA and QB sites. Both quinone sites showed two strongly coupled nitrogen nuclei. However, the hyperfine interactions were greater in magnitude at QA while the QB site showed a larger additional contribution from “matrix” nitrogen. Nitrogen (S = 1) has a quadruple moment and the quadrupole parameters are dependent on the electric field gradient at the nucleus of interest. Using these values, the two strongly coupled nitrogen nuclei were assigned to peptide and histidine for both quinone binding sites. The quadrupole asymmetry parameter was measureable only for the QA site nitrogen. An asymmetry parameter close to ~1 for the histidine nitrogen versus 0.63 for the peptide nitrogen is used to conclude that the histidine has a stronger H-bond than peptide. Mutations to the M265 isoleucine in the QA site to the polar amino acids threonine or serine have shown a significant (80-100mV) change in redox potential versus the wild type. Comparing nitrogen ESEEM from the M265 threonine mutant to the wild type provided insight into these redox potential changes. While nitrogen hyperfine parameters were nearly unchanged between the histidine and the QA semiquinone, changes in the quadrupole parameters indicate a significant change in the electric field gradient. Additionally, resolution of the hyperfine coupling between the peptide nitrogen and the semiquinone is decreased in the mutant. Proton ESEEM showed a 2 exchangeable protons coupled to QA while there were up to 4 coupled to QB. In the QA site these are assigned to histidine at M219 and alanine at M260. In order to assign the protons in the QB site, experimental parameters were compared to ONIOM (QM/MM) calculations. The protons coupled to QB were thus assigned to histidine at L190, peptide protons from L225 and L224 and a serine hydroxyl from L223.

Graduation Semester

2011-05

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http://hdl.handle.net/2142/24256

Copyright and License Information

Copyright 2011 Erik W. Martin

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