Redox intermediates of the Mn-Fe Site in subunit R2 of Chlamydia trachomatis ribonucleotide reductase: an X-ray absorption and EPR study.
The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O(2) activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2(red)) showed an isotropic six-line EPR signal at g approximately 2 of the Mn(II)Fe(II) state. In oxidized R2 (R2(ox)), the Mn(III)Fe(III) state exhibited EPR g values of 2.013, 2.009, and 2.015. By XAS, Mn-Fe distances and oxidation states of intermediates were determined and assigned as follows: approximately 4.15 A, Mn(II)Fe(II); approximately 3.25 A, Mn(III)Fe(II); approximately 2.90 A, Mn(III)Fe(III); and approximately 2.75 A, Mn(IV)Fe(III). Shortening of the Mn/Fe-ligand bond lengths indicated formation of additional metal bridges, i.e. microO(H) and/or peroxidic species, upon O(2) activation at the site. The structural parameters suggest overall configurations of the Mn-Fe site similar to those of homo-metallic sites in other R2 proteins. However, the approximately 2.90 A and approximately 2.75 A Mn-Fe distances, typical for di-microO(H) metal bridging, are shorter than inter-metal distances in any R2 crystal structure. In diffraction data collection, such bridges may be lost due to rapid x-ray photoreduction of high-valent metal ions, as demonstrated here for Fe(III) by XAS.
Voevodskaya
Nina
N
Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden.
Lendzian
Friedhelm
F
Sanganas
Oliver
O
Grundmeier
Alexander
A
Gräslund
Astrid
A
Haumann
Michael
M
D015502:Absorptiometry, Photon; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain / Q000502:physiology; D002692:Chlamydia trachomatis / Q000201:enzymology*; D007501:Iron / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary / Q000502:physiology; D012264:Ribonucleotide Reductases / Q000737:chemistry*
D001426:Bacterial Proteins; D008345:Manganese; D007501:Iron; D012264:Ribonucleotide Reductases