APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.
The hyperthermophilic archaeon Pyrococcus furiosus contains a 4-Fe ferredoxin (Pf-Fd) that differs from most other 4Fe-Fd's in that its [Fed41 cluster is anchored to protein by only three cysteinyl residues. Pf-Fd also is of interest because in its reduced form, [Fe&]+, the cluster exhibits both S = ' / 2 and S = 3/2 spin states. Addition of excess cyanide ion converts the cluster exclusively to an S = '/2 state (gl = 2.09, g2 = 1.95, g3 = 1.92); however, dialysis restores the EPR signal of native reduced protein, indicating that the cluster is not irreversibly altered by cyanide. Both the native protein and protein in the presence of excess cyanide ion (Pf-Fd-CN) were investigated here using the techniques of electron paramagnetic resonance (EPR) and electron-nuclear double-resonance (ENDOR) spectroscopy. No evidence for a strongly coupled solvent-derived hydrogen ('H or 2H) from an OH-or H20 ligand in either spin state of the [Fe4S41f cluster was observed, contrary to an earlier report. Rather, 1,2H ENDOR characteristic of 4Fe-Fd's was seen for both native Pf-Fd and Pf-Fd-CN. Pf-Fd-CN was further investigated using 13CN-and CI5N-ligands. I3C and I5N ENDOR indicated that a single cyanide ion bound directly, with the cluster showing an unusually small contact interaction (aiS0(l3C) --3 MHz, also(15N) -0). This is in contrast to cyanide bound to monomeric low-spin Fe(II1)-containing proteins such as transferrin and myoglobin, for which the I3C hyperfine coupling has a large isotropic component (ais0(13C) % -30 MHz). The full I3C and I5N hyperfine tensors were determined by computer simulation of the ENDOR spectra. The A(I3C) is rotated by -40" about g2. The g and A(13C) tensor information is combined with recently reported single-crystal EPR studies on [Fe4S4]+s3+ model compounds and leads to a simple geometrical picture of cyanide binding to Pf-Fd, in which CN-replaces the Asp-14 ligand and binds in an orientation similar to that of the Cys residue found in an ordinary 4Fe-4S ferredoxin. This reversible binding of an exogenous ligand may have implications for the catalytic activity of Fe-S enzymes.
The electronic and redox properties of the iron and tungsten centers in the aldehyde ferredoxin oxidoreductases (AORs) from Pyrococcus furiosus (Pf) and Pyrococcus strain ES-4 (ES-4) have been investigated by the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Parallel- and perpendicular-mode EPR studies of ES-4 AOR reveal a redox inactive “g = 16” resonance from an integer spin paramagnet. On the basis of the X-ray crystal structure of Pf AOR (Chan, M. K.; Mukund, S.; Kletzin, A.; Adams, M. W. W.; Rees, D. C. Science 1995, 267, 1463−1469), this resonance is attributed to a mononuclear high-spin Fe(II) ion at the subunit interface, although the possibility that this center is a carboxylate-bridged reduced diiron center in ES-4 AOR is also considered. Both enzymes have a [4Fe−4S]2+,+ cluster with unique electronic properties compared to known synthetic or biological [4Fe−4S]+ clusters, i.e. pure S = 3/2 ground state with g = 4.7, 3.4, 1.9 (E/D = 0.12 and D = +4 cm-1). Seven distinct W(V) EPR signals have been observed during dye-mediated redox titrations of Pf AOR, and the four major W(V) species have been rigorously identified and characterized via EPR spectral simulations of natural abundance and 183W-enriched samples (183W, I = 1/2, 14.28% natural abundance). Both enzymes contain two major forms of W, each corresponding to approximately 20−30% of the total W. One of these is a catalytically competent W species that cycles between the W(IV)/W(V)/W(VI) states at physiologically relevant potentials (<−300 mV) and gives rise to the “low-potential” W(V) resonance, g ∼ 1.99, 1.90, 1.86. This form of W is quantitatively and irreversibly converted into a distinct and inactive W(IV)/W(V) species by the addition of high concentrations of glycerol or ethylene glycol at 80 °C and is responsible for the “diol-inhibited” W(V) resonance, g ∼ 1.96, 1.94, 1.89. The other major form of W gives rise to a “high-potential” W(V) species, g ∼ 1.99, 1.96, 1.89, at nonphysiologically relevant potentials (>0 mV), as a result of a one-electron redox process that is tentatively attributed to ligand based oxidation of a W(VI) species. In addition, active samples of Pf AOR, in particular, can have up to 20% of the W as an inactive W(VI)/W(V) species, with a midpoint potential close to −450 mV, and is responsible for the “spin-coupled” W(V) resonance. This W(V) signal exhibits a broad complex resonance spanning 600 mT due to weak spin−spin interaction with the nearby S = 3/2 [4Fe−4S]+ cluster. Structures are proposed for each of the major W(V) species on the basis of EPR g values and 183W A values as compared to other biological and synthetic W(V)/Mo(V) centers, VTMCD spectra, and the available X-ray crystallographic and XAS data for Pf AOR and the Mo-containing DMSO reductase from Rhodobacter sphaeroides. Comparison with the limited spectroscopic data that are available for all known tungstoenzymes suggests two major classes of enzyme with distinct active site structures.
In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H 2 O 2 . However, it is inactivated by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.
Iron, a key element in Fenton chemistry, causes oxygen-related toxicity to cells of most living organisms. Helicobacter pylori is a microaerophilic bacterium that infects human gastric mucosa and causes a series of gastric diseases. Exposure of H. pylori cells to air for 2 h elevated the level of free iron by about 4-fold as measured by electron paramagnetic resonance spectroscopy. H. pylori cells accumulated more free iron as they approached stationary phase growth, and they concomitantly suffered more DNA damage as indicated by DNA fragmentation analysis. Relationships between the intracellular free iron level, specific oxidative stress enzymes, and DNA damage were identified, and new roles for three oxidative stress-combating enzymes in H. pylori are proposed. Mutant cells defective in either catalase (KatA), in superoxide dismutase (SodB) or in alkyl hydroperoxide reductase (AhpC) were more sensitive to oxidative stress conditions; and they accumulated more free (toxic) iron; and they suffered more DNA fragmentation compared to wild type cells. A significant proportion of cells of sodB, ahpC, or katA mutant strains developed into the stress-induced coccoid form or lysed; they also contained significantly higher amounts of 8-oxo-guanine associated with their DNA, compared to wild type cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.