ABSTRACT
Ribonucleotide reductase (RNR) is a key enzyme for DNA synthesis since it provides cells with deoxyribonucleotides, the DNA precursors. Class I alpha2beta2 RNRs contain a dinuclear iron center and an essential tyrosyl radical in the beta2 component (protein R2). This is also true for the purified protein R2 of Mycobacterium tuberculosis RNR, as shown by iron analysis, light absorption and EPR spectroscopy. EPR spectroscopy at 286 GHz revealed a high g(x) value, suggesting that the radical is not hydrogen bonded, as in other prokaryotic R2s and in contrast with eukaryotic R2s (from Arabidopsis thaliana and mouse). Furthermore, it proved to be very resistant to scavenging by a variety of phenols and thiols and by hydroxyurea, similar to the Escherichia coli radical. By comparison, the plant and mouse radicals are very sensitive to drugs such as resveratrol and 2-thiophenthiol. The radical from M. tuberculosis RNR does not seem to be an appropriate target for new antituberculous agents.
Subject(s)
Arabidopsis/enzymology , Escherichia coli/enzymology , Mycobacterium tuberculosis/enzymology , Ribonucleotide Reductases/chemistry , Tyrosine/metabolism , Animals , Bacterial Proteins/chemistry , Electron Spin Resonance Spectroscopy , Free Radical Scavengers/metabolism , Free Radicals/metabolism , Iron/chemistry , Mice , Plant Proteins/chemistry , SpectrophotometryABSTRACT
The photosynthetic reaction center of photosystem I (PS I) contains a phylloquinone molecule (A1) which acts as a transient electron acceptor. In PS I form the cyanobacterium Synechocystis PCC 6803 under reducing conditions, we have photoaccumulated an EPR signal assigned to the phylloquinone radical anion. The phylloquinone EPR spectrum has been studied in oriented multilayers of PS I using EPR at 9 GHz. In addition, the phyllosemiquinone spectrum has been obtained at 283 GHz using high-field, high-frequency EPR spectroscopy. From the orientation dependence of the spectrum at 9 GHz and the resolved g values obtained at 283 GHz, the phyllosemiquinone ring plane was determined to be almost perpendicular to the membrane (76 degrees) while the oxygen-oxygen (O-O) axis of the quinone was found to make an approximate 63 degrees angle to the membrane plane. The orientation of the ring plane is similar to that determined for the quinone electron acceptor (QA) in the purple bacterial reaction center, while the orientation of the O-O axis is significantly different. The new orientation information, when taken with data in the literature, allows the position of the phylloquinone in the reaction center to be better defined.
