Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase

Publication: ResearchPh.D. thesis – Annual report year: 2009

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Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase. / Nørgaard, Hanne; Christensen, Hans Erik Mølager (Supervisor); Ooi, Bee Lean (Supervisor).

Kgs. Lyngby, Denmark : Technical University of Denmark (DTU), 2009. 117 p.

Publication: ResearchPh.D. thesis – Annual report year: 2009

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Author

Nørgaard, Hanne; Christensen, Hans Erik Mølager (Supervisor); Ooi, Bee Lean (Supervisor) / Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase.

Kgs. Lyngby, Denmark : Technical University of Denmark (DTU), 2009. 117 p.

Publication: ResearchPh.D. thesis – Annual report year: 2009

Bibtex

@phdthesis{159d8106c6ce4fe9aac335c4df976929,
title = "Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase",
publisher = "Technical University of Denmark (DTU)",
author = "Hanne Nørgaard and Christensen, {Hans Erik Mølager} and Ooi, {Bee Lean}",
year = "2009",

}

RIS

TY - BOOK

T1 - Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase

A1 - Nørgaard,Hanne

AU - Nørgaard,Hanne

A2 - Christensen,Hans Erik Mølager

A2 - Ooi,Bee Lean

ED - Christensen,Hans Erik Mølager

ED - Ooi,Bee Lean

PB - Technical University of Denmark (DTU)

PY - 2009/11

Y1 - 2009/11

N2 - Part I Two evolutionary old ferredoxins were characterized. LCA Fd – a 55 AA ferredoxin – is believed to have function in the last common ancestor of life on Earth. An internal repeat in the sequence of LCA Fd was phylogenetically traced even further back in evolution resulting in a 23 AA peptide named Ori-ISP – origin of iron-sulfur proteins. It was found that LCA ferredoxin could exist in at least two forms: LCA Fd1 and LCA Fd2. LCA Fd1 was studied using cyclic voltammetry. Two signals were observed in the voltammograms; one strong signal from [4Fe-4S] clusters at -430 – -415 mV vs. NHE and a significantly weaker signal probably from a small contribution of [3Fe-4S] at 47 mV vs. NHE. EPR-monitored redox titration showed one major transition at -345 mV vs. NHE. X-band EPR spectra from the titration revealed spectral changes depending on degree of reduction, and a complex spectrum was observed for the fully reduced LCA Fd1. Furthermore a signal at half-field was observed for the fully reduced sample. The EPR results were interpreted as being caused by two dipole-dipole interacting [4Fe-4S] clusters in the protein. A Q-band EPR spectrum showed that the field independent dipoledipole interaction was relatively weaker at these conditions. Simulation of partly reduced LCA Fd1 X-band EPR spectrum resulted in g = [2.065; 1.926; 1.88]. Mass spectrometry confirmed that LCA Fd1 contains two [4Fe-4S] clusters. LCA Fd2 was also studied using EPR-monitored redox titration. The X-band EPR spectra showed a weakly anisotropic feature (g = [2.008; 2.008; 1.99]) which disappeared upon reduction (E = - 45 mV vs. NHE). At lower potentials (E = -350 mV vs. NHE) an anisotropic spectrum (g = [2.062; 1.93; 1.88]) was observed. Based on the EPR spectroscopic data it was suggested that LCA Fd2 contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Reconstituted Ori-ISP was found to be very oxygen sensitive. Characterizing the Ori-ISP using cyclic voltammetry proved to be very difficult, most probably because the protein was not stable under the conditions used for measurement. EPR-monitored redox titration showed one major redox transition at -305 mV vs. NHE. X-band EPR spectra proved to be complicated probably due to dipoledipole interaction between clusters, however no half-field signal was observed. At Q-band conditions the dipole-dipole interaction was not observed and the data could be fitted with a [4Fe-4S] cluster with g = [2.075; 1.92; 1.90]. These findings suggest that Ori-ISP forms dimers upon reconstitution coordinating two [4Fe-4S] clusters per dimer in a structure similar to that of LCA Fd1. iv Part II Class I and class Ic ribonucleotide reductases (RNR) are very similar in function and structure. Major differences are that the R2 subunit in class I RNR function using a tyrosyl radical located close to a [Fe-Fe] site, while class Ic RNR function with a [Mn-Fe] site. In class Ic RNR a phenylalanine is found where the tyrosyl radical is formed in class I RNR. Chlamydia trachomatis (Ct) RNR belongs to class Ic, and the variant CtR2 F127Y was investigated. In the variant the tyrosine from conventional class I RNR was introduced in CtR2. Activity measurements of CtR2 F127Y showed that the variant could be activated by O2, but that the activity was lost after ~1 hour incubation at ambient conditions, whereas CtR2 WT did not lose activity under the same conditions. Freeze-quenched EPR used to investigate to oxygen activation of CtR2 F127Y [MnII-FeII] showed that transient [MnIV-FeIV] was formed prior to the active form (most likely [MnIV-FeIII]). EPR spectra of the product upon reaction of CtR2 F127Y [MnII-FeII] with oxygen for extended periods of time (> two days) showed that the cofactor was found in the inactive [MnIII-FeIII] state.

AB - Part I Two evolutionary old ferredoxins were characterized. LCA Fd – a 55 AA ferredoxin – is believed to have function in the last common ancestor of life on Earth. An internal repeat in the sequence of LCA Fd was phylogenetically traced even further back in evolution resulting in a 23 AA peptide named Ori-ISP – origin of iron-sulfur proteins. It was found that LCA ferredoxin could exist in at least two forms: LCA Fd1 and LCA Fd2. LCA Fd1 was studied using cyclic voltammetry. Two signals were observed in the voltammograms; one strong signal from [4Fe-4S] clusters at -430 – -415 mV vs. NHE and a significantly weaker signal probably from a small contribution of [3Fe-4S] at 47 mV vs. NHE. EPR-monitored redox titration showed one major transition at -345 mV vs. NHE. X-band EPR spectra from the titration revealed spectral changes depending on degree of reduction, and a complex spectrum was observed for the fully reduced LCA Fd1. Furthermore a signal at half-field was observed for the fully reduced sample. The EPR results were interpreted as being caused by two dipole-dipole interacting [4Fe-4S] clusters in the protein. A Q-band EPR spectrum showed that the field independent dipoledipole interaction was relatively weaker at these conditions. Simulation of partly reduced LCA Fd1 X-band EPR spectrum resulted in g = [2.065; 1.926; 1.88]. Mass spectrometry confirmed that LCA Fd1 contains two [4Fe-4S] clusters. LCA Fd2 was also studied using EPR-monitored redox titration. The X-band EPR spectra showed a weakly anisotropic feature (g = [2.008; 2.008; 1.99]) which disappeared upon reduction (E = - 45 mV vs. NHE). At lower potentials (E = -350 mV vs. NHE) an anisotropic spectrum (g = [2.062; 1.93; 1.88]) was observed. Based on the EPR spectroscopic data it was suggested that LCA Fd2 contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Reconstituted Ori-ISP was found to be very oxygen sensitive. Characterizing the Ori-ISP using cyclic voltammetry proved to be very difficult, most probably because the protein was not stable under the conditions used for measurement. EPR-monitored redox titration showed one major redox transition at -305 mV vs. NHE. X-band EPR spectra proved to be complicated probably due to dipoledipole interaction between clusters, however no half-field signal was observed. At Q-band conditions the dipole-dipole interaction was not observed and the data could be fitted with a [4Fe-4S] cluster with g = [2.075; 1.92; 1.90]. These findings suggest that Ori-ISP forms dimers upon reconstitution coordinating two [4Fe-4S] clusters per dimer in a structure similar to that of LCA Fd1. iv Part II Class I and class Ic ribonucleotide reductases (RNR) are very similar in function and structure. Major differences are that the R2 subunit in class I RNR function using a tyrosyl radical located close to a [Fe-Fe] site, while class Ic RNR function with a [Mn-Fe] site. In class Ic RNR a phenylalanine is found where the tyrosyl radical is formed in class I RNR. Chlamydia trachomatis (Ct) RNR belongs to class Ic, and the variant CtR2 F127Y was investigated. In the variant the tyrosine from conventional class I RNR was introduced in CtR2. Activity measurements of CtR2 F127Y showed that the variant could be activated by O2, but that the activity was lost after ~1 hour incubation at ambient conditions, whereas CtR2 WT did not lose activity under the same conditions. Freeze-quenched EPR used to investigate to oxygen activation of CtR2 F127Y [MnII-FeII] showed that transient [MnIV-FeIV] was formed prior to the active form (most likely [MnIV-FeIII]). EPR spectra of the product upon reaction of CtR2 F127Y [MnII-FeII] with oxygen for extended periods of time (> two days) showed that the cofactor was found in the inactive [MnIII-FeIII] state.

BT - Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase

ER -