Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the presence of ascorbic acid, the reductive-independent 59Fe uptake route is investigated. On the other hand, in the absence of ascorbic acid, the reductive-dependent 59Fe uptake route is stimulated. Using this strategy for the human pathogenic fungus Paracoccidioides species, the results showed that iron uptake by Pb01 in the absence of ascorbic acid was low, unlike what was observed for Pb18. These results suggest that only in Pb18 the iron uptake pathway is coupled to a ferric reductase (Bailão et al., 2015). In this protocol, we describe how to perform 59Fe uptake assays in Paracoccidioides species.
Saccharomyces cerevisiae, which lack a functional SOD1 gene, encoding the cytosolic Cu,Zn-superoxide dismutase (SOD1), exhibit a variety of metabolic defects in aerobic but not in anaerobic growth. We test here the hypothesis that some of these defects may be due to specific transcriptional changes programmed for cell survival under dioxygen stress. Analysis of the budding pattern and generation time showed that the slower proliferation of an sod1Δ mutant strain under air was due to an increase from 42 to 89 min spent in the G1 phase of the cell cycle. This delay in G1 was not due to an overall decline in biosynthetic activity since total protein and mRNA synthesis was not reduced even under 100% O2. However, rRNA synthesis was strongly decreased, e.g. by 80% in the mutant under 100% O2 (in comparison to N2). Under these conditions, the mutant permanently arrested in G1; this arrest was due to an inhibition of the Start function that prepares yeast for S phase. This Start arrest was due to an inhibition of transcription of the autoregulated G1 cyclins, CLN1 and CLN2; the transcription of the constitutive G1 cyclin, CLN3, was unaffected by the stress. Expression of a hyperstable Cln3 prevented the G1 arrest, indicating that it was due solely to the inhibition of cell cycle-dependent cyclin expression. This remodeling of transcription in oxidative stress was seen also in the inhibition of glucose derepression of SUC2 expression. In contrast, the signaling and activation of mating pheromone (FUS1) and copper-responsive (CUP1) promoter activity were not affected by dioxygen stress, while genes encoding other anti-oxidant enzymes (SOD2, CTT1 and CTA1) were strongly induced. The UBI loci, encoding ubiquitin, were particularly good examples of this pattern of negative and positive transcriptional response to the stress. UBI1-UBI3 expression was repressed in the mutant under 100% O2, while expression of UBI4 was strongly induced. The data demonstrate that extensive remodeling of transcription occurs in yeast under a strong dioxygen stress. This remodeling results in a pattern of expression of gene products needed for defense and repair, and suppression of activities associated with normal proliferative growth. Saccharomyces cerevisiae, which lack a functional SOD1 gene, encoding the cytosolic Cu,Zn-superoxide dismutase (SOD1), exhibit a variety of metabolic defects in aerobic but not in anaerobic growth. We test here the hypothesis that some of these defects may be due to specific transcriptional changes programmed for cell survival under dioxygen stress. Analysis of the budding pattern and generation time showed that the slower proliferation of an sod1Δ mutant strain under air was due to an increase from 42 to 89 min spent in the G1 phase of the cell cycle. This delay in G1 was not due to an overall decline in biosynthetic activity since total protein and mRNA synthesis was not reduced even under 100% O2. However, rRNA synthesis was strongly decreased, e.g. by 80% in the mutant under 100% O2 (in comparison to N2). Under these conditions, the mutant permanently arrested in G1; this arrest was due to an inhibition of the Start function that prepares yeast for S phase. This Start arrest was due to an inhibition of transcription of the autoregulated G1 cyclins, CLN1 and CLN2; the transcription of the constitutive G1 cyclin, CLN3, was unaffected by the stress. Expression of a hyperstable Cln3 prevented the G1 arrest, indicating that it was due solely to the inhibition of cell cycle-dependent cyclin expression. This remodeling of transcription in oxidative stress was seen also in the inhibition of glucose derepression of SUC2 expression. In contrast, the signaling and activation of mating pheromone (FUS1) and copper-responsive (CUP1) promoter activity were not affected by dioxygen stress, while genes encoding other anti-oxidant enzymes (SOD2, CTT1 and CTA1) were strongly induced. The UBI loci, encoding ubiquitin, were particularly good examples of this pattern of negative and positive transcriptional response to the stress. UBI1-UBI3 expression was repressed in the mutant under 100% O2, while expression of UBI4 was strongly induced. The data demonstrate that extensive remodeling of transcription occurs in yeast under a strong dioxygen stress. This remodeling results in a pattern of expression of gene products needed for defense and repair, and suppression of activities associated with normal proliferative growth.
Multicopper oxidases (MCOs) carry out the most energy efficient reduction of O2 to H2O known, i.e., with the lowest overpotential. This four-electron process requires an electron mediating type 1 (T1) Cu site and an oxygen reducing trinuclear Cu cluster (TNC), consisting of a binuclear type 3 (T3)- and a mononuclear type 2 (T2) Cu center. The rate-determining step in O2 reduction is the first two-electron transfer from one of the T3 Cu's (T3β) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3β Cu variants of the Fet3p, where a first shell His ligand is mutated to Glu or Gln. This converts the fast two-electron reaction of the wild-type (WT) enzyme to a slow one-electron oxidation of the TNC. Both variants initially react to form a common T3β Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3β Cu in the reduced TNCs in the two variants, in contrast to the trigonal arrangement observed in the WT enzyme. This structural perturbation is found to significantly alter the electronic structure of the reduced TNC, which is no longer capable of rapidly transferring two electrons to the two perpendicular half occupied π*-orbitals of O2, in contrast to the WT enzyme. This study provides new insight into the geometric and electronic structure requirements of a fully functional TNC for the rate determining two-electron reduction of O2 in the MCOs.
Abstract Kopplungskonstanten der starren Moleküle des Semichinons (I) bzw. dessen Dideuteroderivates und des Radikalanions (II), bei denen der β‐ständige F‐ Kern bezüglich der Knotenebene des π‐Elektronensystems festgelegt ist, werden bestimmt, um zu klären bei welchen Winkeln EPR‐Kopplungskonstanten β‐ständiger Fluor‐Substituenten in freien Radikalen am größten sind.
