Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

The [2Fe–2S] transcriptional factor SoxR, a member of the MerR family, functions as a sensor of oxidative stress in <i>Escherichia coli</i>. The transcriptional activity of SoxR is regulated by the reversible oxidation and reduction of [2Fe–2S] clusters. Electrochemistry measurements on DNA-modified electrodes have shown a dramatic shift in the reduction potential of SoxR from −290 to +200 mV with the promoter DNA-bound [Gorodetsky, A. A., Dietrich, L. E. P., Lee, P. E., Demple, B., Newman, D. K., and Barton, J. K. (2008) DNA binding shifts the reduction potential of the transcription factor SoxR, Proc. Natl. Acad. Sci. U.S.A. 105, 3684−3689]. To determine the change of the SoxR reduction potential using the new condition, the one-electron oxidation–reduction properties of [2Fe–2S] cluster in SoxR were investigated in the absence and presence of the DNA. The [2Fe–2S] cluster of SoxR was completely reduced by nicotinamide adenine dinucleotide phosphate (NADPH)–cytochrome P450 reductase (CRP) in the presence of a NADPH generating system (glucose 6-dehydrogenase and glucose-6 phosphate), indicating that CRP can serve as an NADPH-dependent electron carrier for SoxR. The reduction potential of SoxR was measured from equilibrium data coupled with NADPH and CRP in the presence of electron mediators. The reduction potentials of DNA-bound and DNA-free states of SoxR were −320 and −293 mV versus NHE (normal hydrogen electrode), respectively. These results indicate that DNA binding causes a moderate shift in the reduction potential of SoxR

Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues

In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O₂^•–) has a direct role as a signal for <i>E. coli</i> SoxR and that the sensitivity of the <i>E. coli</i> SoxR response to O₂^•– is 10-fold higher than that of <i>Pseudomonas aeruginosa</i> SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O₂^•– activation. To investigate the determinants of SoxR’s sensitivity to O₂^•–, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O₂^•– using pulse radiolysis. The substitution of <i>E. coli</i> SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein’s reaction with O₂^•–. The second-order rate constant of the reaction was 3.3 × 10⁷ M^–1 s^–1, which was 10 times smaller than that of wild-type SoxR. Conversely, the corresponding substitution of Ala90 with Lys in <i>P. aeruginosa</i> SoxR increased the rate approximately 10-fold. In contrast, introductions of the Arg127Ser128Asp129 → Leu127Gln128Ala129 substitution into <i>E. coli</i> SoxR, and the corresponding substitution (Leu125Gln126Ala127 → Arg125Ser126Asp127) in <i>P. aeruginosa</i> SoxR, did not affect the reaction rates. In addition, the Lys mutation in <i>E. coli</i> SoxR (K89AK92A) showed a defect in vivo transcriptional activity by measuring β-galactosidase expression in response to paraquat. Our findings clearly support the idea Lys is critical to the response to O₂^•– and further transcriptional activity of SoxR

Reaction Intermediates of Nitric Oxide Synthase from <i>Deinococcus radiodurans</i> as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex

Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate <i>N</i>-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from <i>Deinococcus radiodurans</i> was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs. Subsequently, ferrous heme reacted with O₂ to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 10⁸ M^–1 s^–1. In the tetrahydrofolate (H₄F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 10³ s^–1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H₄F, this step did not proceed, suggesting that H₄F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s^–1. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s^–1). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates

Dynamics of Radical Ions of Hydroxyhexafluoroisopropyl-Substituted Benzenes

Fluorination of resist materials is an effective method used to enhance the energy deposition of extreme ultraviolet (EUV) light in the fabrication of next-generation semiconductor devices. The dynamics of radical ions are important to understand when considering the radiation-chemistry of the resist materials using EUV and electron beam lithography. Here, the dynamics of the radical anions and cations of benzenes with one or two 2-hydroxyhexafluoroisopropyl groups (HFABs) were studied using radiolysis techniques. The formation of dimer radical cations was observed only in the monosubstituted benzene solutions of 1,2-dichloroethane. If the compound contained more than two substituents, it was found to hinder the necessary π–π overlapping. Pulse radiolysis of HFABs in tetrahydrofuran showed a characteristic spectral shift of the radical anion within the region of several hundred nanoseconds. From the results of low-temperature spectroscopy and density functional calculations, it is suggested that excess electrons of the 2-hydroxyhexafluoroisopropyl group of the radical anions cause dissociation into neutral radicals