Structure and function studies of polar mutants of the QA pocket in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides

Mattis, Aidas Joseph

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Description

Title

Structure and function studies of polar mutants of the QA pocket in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides

Author(s)

Mattis, Aidas Joseph

Issue Date

2016-04-15

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

Crofts, Anthony R.

Doctoral Committee Chair(s)

Crofts, Anthony R.

Committee Member(s)

• Gennis, Robert B.
• Kranz, David M.
• Nair, Satish K.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Reaction Center
• X-ray Crystal Structure

Abstract

The bacterial photosynthetic reaction center (RC) is the protein that converts light to chemical energy. The light is initially absorbed by a pair of bacteriochlorophylls that then transfer an electron through a series of cofactors until it reaches the final two electron acceptors, the primary quinone (QA) that then reduce the secondary quinone (QB). In Rb. sphaeroides these quinones are chemically identical ubiquinones and the protein must tune the midpoint potential (Em) of each quinone to make electron transfer from QA to QB favorable. Using site directed mutagenesis together with the techniques of X-ray crystallography, flash kinetic spectroscopy, and quinone substitution, I was able to probe how structural changes contribute to Em changes and work to better understand the physical chemistry involved. The mutation of the wild type (WT) Ile at the 265th amino acid of the M subunit (M265), which lies within van der Waals contact to the primary quinone (QA), to the polar hydroxyl (O-H) mutants of Ser (M265IS) and Thr (M265IT), previously showed a drop in the in situ Em of QA by 85 and 100 mV, respectively (Takahashi et al 2001). In repeating Takahashi et al’s kinetic work, it was discovered that there are two separate components for the QA- back reaction not previously recognized. The structures of the two mutants were solved using X-ray crystallography and the orientation of the M265 side chain O-H, relative to the quinone, for the two mutants are in different orientations. The M265IS O-H is located in a position where four potential hydrogen bonds (H-bonds) are present, while the M265IT O-H is positioned where the O-H has only one potential H-bond. QA in M265IS has an additional H-bond, not present in WT, between the 2-methoxy of QA and the backbone nitrogen of M249 that maybe necessary to stabilize the quinone due to the increase in the size of the quinone binding pocket. For both hydroxyl mutants the H-bond to the C1 carbonyl of QA was significantly shorter than Xray-avg (the average of all atomic distances from currently deposited RC X-ray structures with resolution better than 2.80 Å) while only the H-bond to the C4 carbonyl of QA from M265IT was significantly shorter. The Ile at M265 was also mutated to the polar amide mutants Asn (M265IN) and Gln (M265IQ). M265IN presented kinetics not very different to M265IT, indicating that the in situ Em of QA was similar. However, M265IQ showed a slower QA- back reaction, which is opposite from the other three polar mutants. Both mutants showed two component kinetics for the QA- back reaction that varied with pH. The QB- back reaction was also slower for M265IQ compared to WT, which is the same direction as the other mutants. These results indicate that the in situ Em of M265IQ is likely unchanged from WT. It was further found that QA of M265IQ was only occupied approximately 50% of the time. The structures of M265IN and M265IQ were solved using X-ray crystallography. M265IN showed that the side chain only took on one conformation, but the rotamer of the side chain amide could possibly take on two orientations. M265IQ showed two conformations for the side chain of M265 consistent with one conformation of QA bound (Conf. A) and the other with QA dissociated (Conf. B) or bound at a more distant site from the WT binding position. The amine and carbonyl of the side chain of Asn-M265 showed both H-bond and repulsion with either the C4 carbonyl of QA or the δ nitrogen of His-M219 depending on the rotamer of the amide. The side chain amine of Conf. A of M265IQ has an internal H-bond with the backbone carbonyl and the side chain carbonyl has a potential H-bond to the δ nitrogen of His-M219, which bifurcates the δ nitrogen H-bond between the side chain of M265 and the C4 carbonyl of the quinone. Both mutants showed longer H-bonds between the C4 quinone carbonyl and δ nitrogen of His-M219 when compared to Xray-avg, but only M265IQ mutant showed a shorter hydrogen bond between the C1 quinone carbonyl and the M260 backbone N. The M265IN C1 quinone carbonyl H-bond was not significantly different from Xray-avg. The RC is a finely tuned system that tightly controls the midpoint potentials of QA and QB so that an electron can be favorably transferred from one ubiquinone to another. The addition of a polar group to the non-polar QA site decreases the midpoint potential by approximately 100 mV for M265IS, M265IT, and M265IN. However, M265IQ is such a structurally large amino acid addition to the RC that the quinone is displaced 50% of the time from its WT location and gives a much more complicated kinetic picture. Based on the crystal structures of M265IS and M265IT, the orientation of the hydroxyl controls the Em, but to a much smaller extent than simply the addition of the polar group to the local vicinity of the QA site. The addition of an amine group to QA has a similar Em change to the addition of a hydroxyl. I therefore conclude that local electrostatics are likely the largest factor in controlling the Em of QA. Electrostatic calculations are needed to calculate how adding a polar group at M265 changes the Em of QA.

Graduation Semester

2016-05

Type of Resource

text

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Copyright 2016 Aidas J Mattis

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