Probing the mechanism of conformationally gated inter-subunit radical transfer in the Mn/Fe-dependent ribonucleotide reductase from Chlamydia trachomatis
Open Access
Author:
Xie, Jiajia
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
October 07, 2008
Committee Members:
Joseph M Bollinger Jr., Thesis Advisor/Co-Advisor
Keywords:
PCET ribonucleotide reductase
Abstract:
A conventional class I ribonucleotide reductase (RNR) employs a diferric-tyrosyl radical (Y•) cofactor in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from Chlamydia trachomatis (Ct), uses a Mn(IV)/Fe(III) cofactor in place of the Y• for radical initiation. The residues for PCET are thought to be conserved in both systems. The properties of the PCET in Ct provided a unique chance to study the mechanism of conformationally gated inter-subunit PCET in Ct RNR. In this study, site-directed mutation showed resides W51, Y338 in R2, Y991, Y990 and C672 in R1 are essential for PCET and without any of them could almost totally inactivate the enzyme. We showed the pathway radicals were accumulated by formation of the one-electron more oxidized Mn(IV)/Fe(IV) form of the cofactor in the presence of R1 and substrate within variant proteins. We also used sodium dithionite as a reductant to test its ability to reduce the active form Mn(IV)/Fe(III) to Mn(III)/Fe(III) and produce a well-resolved EPR signal. The well-resolved EPR signal is due to the binding of R1 (CDP) to Mn(III)/Fe(III)–R2 causing a conformational change that is propagated to the cofactor in R2. From our results, residues W51, Y338 in R2, Y991, Y990 and C672 play no role in this conformational change behavior. Additionally, we also studied the mechanism of the radical scavenger-hydroxyurea (HU) reduction of Mn(IV)/Fe(III) cluster. Combination of the EPR and stopped-flow experiments, W51-Y222 and W51-Y338 pathways can be two candidates to direct HU reduction.
