Influence of the Chelation Process on the Stability of Organic Trace Mineral Supplements Used in Animal Nutrition.

The effect of the chelation process on the pH-dependent stability of organic trace minerals (OTMs) used as mineral supplements in animal nutrition was assessed using analytical techniques such as potentiometry, Fourier Transform Infrared Spectroscopy (FTIRS) and amino acid profiling. The aim was to understand the influence and relative importance of the manufacturing conditions on mineral chelation and the subsequent pH stability of OTMs. A selection of OTMs were assessed over a wide pH range to account for the typical environmental changes encountered in the gastrointestinal (GI) tract. In the case of proteinate type products, the potentiometric assessment of free mineral concentration indicated that the hydrolysis procedure used to generate the chelating peptides was the major influencer of the pH stability of the products. Many products are available under the umbrella term "OTMs", including amino acid complexes, amino acid chelates, polysaccharide complexes and proteinates. Significant differences in the pH-dependent stability of a range of commercially available OTMs were observed.

Carbon Catabolite Repression Governs Diverse Physiological Processes and Development in Aspergillus nidulans.

Carbon catabolite repression (CCR) is a common phenomenon of microorganisms that enable efficient utilization of carbon nutrients, critical for the fitness of microorganisms in the wild and for pathogenic species to cause infection. In most filamentous fungal species, the conserved transcription factor CreA/Cre1 mediates CCR. Previous studies demonstrated a primary function for CreA/Cre1 in carbon metabolism; however, the phenotype of creA/cre1 mutants indicated broader roles. The global function and regulatory mechanism of this wide-domain transcription factor has remained elusive. Here, we applied two powerful genomics methods (transcriptome sequencing and chromatin immunoprecipitation sequencing) to delineate the direct and indirect roles of Aspergillus nidulans CreA across diverse physiological processes, including secondary metabolism, iron homeostasis, oxidative stress response, development, N-glycan biosynthesis, unfolded protein response, and nutrient and ion transport. The results indicate intricate connections between the regulation of carbon metabolism and diverse cellular functions. Moreover, our work also provides key mechanistic insights into CreA regulation and identifies CreA as a master regulator controlling many transcription factors of different regulatory networks. The discoveries for this highly conserved transcriptional regulator in a model fungus have important implications for CCR in related pathogenic and industrial species. IMPORTANCE The ability to scavenge and use a wide range of nutrients for growth is crucial for microorganisms' survival in the wild. Carbon catabolite repression (CCR) is a transcriptional regulatory phenomenon of both bacteria and fungi to coordinate the expression of genes required for preferential utilization of carbon sources. Since carbon metabolism is essential for growth, CCR is central to the fitness of microorganisms. In filamentous fungi, CCR is mediated by the conserved transcription factor CreA/Cre1, whose function in carbon metabolism has been well established. However, the global roles and regulatory mechanism of CreA/Cre1 are poorly defined. This study uncovers the direct and indirect functions of CreA in the model organism Aspergillus nidulans over diverse physiological processes and development and provides mechanistic insights into how CreA controls different regulatory networks. The work also reveals an interesting functional divergence between filamentous fungal and yeast CreA/Cre1 orthologues.

Quantitative assessment of copper proteinates used as animal feed additives using ATR-FTIR spectroscopy and powder X-ray diffraction (PXRD) analysis.

The aim of the present work was to establish a reliable analytical method to determine the degree of complexation in commercial metal proteinates used as feed additives in the solid state. Two complementary techniques were developed. Firstly, a quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic method investigated modifications in vibrational absorption bands of the ligand on complex formation. Secondly, a powder X-ray diffraction (PXRD) method to quantify the amount of crystalline material in the proteinate product was developed. These methods were developed in tandem and cross-validated with each other. Multivariate analysis (MVA) was used to develop validated calibration and prediction models. The FTIR and PXRD calibrations showed excellent linearity (R2 > 0.99). The diagnostic model parameters showed that the FTIR and PXRD methods were robust with a root mean square error of calibration RMSEC ≤3.39% and a root mean square error of prediction RMSEP ≤7.17% respectively. Comparative statistics show excellent agreement between the MVA packages assessed and between the FTIR and PXRD methods. The methods can be used to determine the degree of complexation in complexes of both protein hydrolysates and pure amino acids.

Synthesis of New Benzocyclotrimer Analogues: New Receptors for Tetramethylammonium Ion Recognition.

Using a [2 + 2 + 2] cycloaddition/Mitsunobu reaction sequence, a convenient synthesis to access new benzocyclotrimer analogues has been developed. The new receptors have the geometry and functionality capable of recognizing the tetramethylammonium ion in the gas phase and in solution.

Dual DNA binding and coactivator functions of Aspergillus nidulans†TamA, a Zn(II)2Cys6 transcription factor.

Transcription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulans TamA and the related Saccharomyces cerevisiae Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function. TamA acts at several promoters as a coactivator of the global nitrogen GATA transcription factor AreA. We now show that TamA is the major transcriptional activator of gdhA, encoding the key nitrogen metabolism enzyme NADP-glutamate dehydrogenase. Moreover, activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. We show that the TamA DNA binding motif is required for DNA binding of FLAG-epitope-tagged TamA to the gdhA promoter. We identify a conserved promoter element required for TamA activation, and show that TamA and AreA are reciprocally required for full binding at the gdhA promoter under conditions where AreA is inactive at most promoters but active at gdhA. Therefore TamA has dual functions as a DNA-binding transcription factor and a non-DNA-binding coactivator. Dual DNA-binding and coactivator functions provide an additional level of combinatorial control to mediate gene-specific expression.

Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.

The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.

HgrA is necessary and sufficient to drive hyphal growth in the dimorphic pathogen Penicillium marneffei.

Fungi produce multiple morphological forms as part of developmental programs or in response to changing, often stressful, environmental conditions. An opportunistic pathogen of humans, Penicillium marneffei displays multicellular hyphal growth and asexual development (conidiation) in the environment at 25°C and unicellular yeast growth in macrophages at 37°C. We characterized the transcription factor, hgrA, which contains a C(2)H(2) DNA binding domain closely related to that of the stress-response regulators Msn2/4 of Saccharomyces cerevisiae. Northern hybridization analysis demonstrated that hgrA expression is specific to hyphal growth, and its constitutive overexpression prevents conidiation and yeast growth, even in the presence of inductive cues, and causes apical hyperbranching during hyphal growth. Consistent with its expression pattern, deletion of hgrA causes defects in hyphal morphogenesis and the dimorphic transition from yeast cells to hyphae. Specifically, loss of HgrA causes cell wall defects, reduced expression of cell wall biosynthetic enzymes and increased sensitvity to cell wall, oxidative, but not osmotic stress agents. These data suggest that HgrA does not have a direct role in the response to stress but is an inducer of the hyphal growth program and its activity must be downregulated to allow alternative developmental programs, including the morphogenesis of yeast cells in macrophages.

Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans.

The ability of fungi to use carbon sources metabolized via the TCA cycle requires gluconeogenesis. In Aspergillus nidulans the AcuK and AcuM transcription factors regulate the expression of the gluconeogenic genes acuF, encoding phosphoenolpyruvate carboxykinase, and acuG, encoding fructose-1,6-bisphosphatase. Expressed proteins containing the AcuK/AcuM N-terminal DNA-binding domains bind together in vitro to motifs containing repeats of CGG separated by seven bases (CCGN7CCG) and the functionality of these sequences was verified in vivo by acuF-lacZ reporter studies. Chromatin immunoprecipitation analysis showed inter-dependent DNA binding of the proteins to the promoters of gluconeogenic genes in vivo independent of the carbon source. Deletion of the mdhC gene encoding a cytoplasmic/peroxisomal malate dehydrogenase showed that this activity is not essential for gluconeogenesis and indicated that induction of AcuK/AcuM regulated genes might result from malate accumulation. Deletion of the gene for the alternative oxidase did not affect growth on gluconeogenic carbon sources; however, expression was absolutely dependent on AcuK and AcuM. Orthologues of AcuK and AcuM, are present in a wide range of fungal taxa and the CCGN7CCG motif is present in the 5' of many genes involved in gluconeogenesis indicating a fundamental role for these transcription factors in reprogramming fungal carbon metabolism.

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei.

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.

Role of carnitine acetyltransferases in acetyl coenzyme A metabolism in Aspergillus nidulans.

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while ß-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm.

Mechanisms of hydrolysis of phenyl- and benzyl 4-nitrophenyl-sulfamate esters.

The kinetics of hydrolysis at medium acid strength (pH interval 2-5) of a series of phenylsulfamate esters 1 have been studied and they have been found to react by an associative S(N)2(S) mechanism with water acting as a nucleophile attacking at sulfur, cleaving the S-O bond with simultaneous formation of a new S-O bond to the oxygen of a water molecule leading to sulfamic acid and phenol as products. In neutral to moderate alkaline solution (pH ≥ ~ 6-9) a dissociative (E1cB) route is followed that involves i) ionization of the amino group followed by ii) unimolecular expulsion of the leaving group from the ionized ester to give N-sulfonylamine [HN=SO(2)] as an intermediate. In more alkaline solution further ionization of the conjugate base of the ester occurs to give a dianionic species which expels the aryloxide leaving group to yield the novel N-sulfonylamine anion [(-)N=SO(2)]; in a final step, rapid attack of hydroxide ion or a water molecule on it leads again to sulfamic acid. A series of substituted benzyl 4-nitrophenylsulfamate esters 4 were hydrolysed in the pH range 6.4-14, giving rise to a Hammett relationship whose reaction constant is shown to be consistent with the E1cB mechanism.

Ga(III) complexes as models for the M(III) site of purple acid phosphatase: ligand effects on the hydrolytic reactivity toward bis(2,4-dinitrophenyl) phosphate.

The effects of a series of Ga(III) complexes with tripodal ligands on the hydrolysis rate of the activated phosphate diester bis(2,4-dinitrophenyl)phosphate (BDNPP) have been investigated. In particular, the influence of the nature of the ligand donor sites on the reactivity of Ga(III) which represents a mimic of the Fe(III) ion in purple acid phosphatase has been evaluated. It has been shown that replacing neutral nitrogen donor atoms and carboxylate groups by phenolate groups enhanced the reactivity of the Ga complexes. Bell-shaped pH-rate profiles and the measured solvent deuterium isotope effects are indicative of a mechanism that involves nucleophilic attack on the coordinated substrate by Ga-OH. The trend in reactivity found for the different Ga complexes reveals that of the two effects of the metal, Lewis acid activation of the substrate and nucleophile activation, the latter one is more important in determining the intrinsic reactivity of the metal catalyst. The relevance of the present findings for the modulation of the activity of the M(III) ion in purple acid phosphatase whose active site contains a phenolate (tyrosine side chain) is discussed.

ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules. A source of cytoplasmic acetyl-CoA is essential for the production of fatty acids and sterols and for protein acetylation, including histone acetylation in the nucleus. In Saccharomyces cerevisiae and Candida albicans acetyl-CoA is produced from acetate by cytoplasmic acetyl-CoA synthetase, while in plants and animals acetyl-CoA is derived from citrate via ATP-citrate lyase. In the filamentous ascomycete Aspergillus nidulans, tandem divergently transcribed genes (aclA and aclB) encode the subunits of ATP-citrate lyase, and we have deleted these genes. Growth is greatly diminished on carbon sources that do not result in cytoplasmic acetyl-CoA, such as glucose and proline, while growth is not affected on carbon sources that result in the production of cytoplasmic acetyl-CoA, such as acetate and ethanol. Addition of acetate restores growth on glucose or proline, and this is dependent on facA, which encodes cytoplasmic acetyl-CoA synthetase, but not on the regulatory gene facB. Transcription of aclA and aclB is repressed by growth on acetate or ethanol. Loss of ATP-citrate lyase results in severe developmental effects, with the production of asexual spores (conidia) being greatly reduced and a complete absence of sexual development. This is in contrast to Sordaria macrospora, in which fruiting body formation is initiated but maturation is defective in an ATP-citrate lyase mutant. Addition of acetate does not repair these defects, indicating a specific requirement for high levels of cytoplasmic acetyl-CoA during differentiation. Complementation in heterokaryons between aclA and aclB deletions for all phenotypes indicates that the tandem gene arrangement is not essential.

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation.

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial beta-oxidation pathways. Prior to channelling the fatty acids into beta-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.

Deletion of the RING-finger peroxin 2 gene in Aspergillus nidulans does not affect meiotic development.

Peroxins are required for protein import into peroxisomes as well as for peroxisome biogenesis and proliferation. Loss-of-function mutations in genes for the RING-finger peroxins Pex2, Pex10 and Pex12 lead to a specific block in meiosis in the ascomycete Podospora anserina. However, loss of protein import into peroxisomes does not result in this meiotic defect. Therefore, it has been suggested that these peroxins have a specific function required for meiosis. To determine whether this role is conserved in other filamentous fungi, we have deleted the gene encoding Pex2 in Aspergillus nidulans. The phenotypes resulting from this deletion are no different from those of previously isolated pex mutants affected in peroxisomal protein import, and viable ascospores are produced in selfed crosses. Therefore, the role of the RING-finger peroxins in meiosis is not conserved in filamentous ascomycetes.

The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were unsuccessful, but we did isolate an rfxA(+)/rfxADelta heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25 degrees C and 37 degrees C. This was also observed for the heterozygous diploid strain during growth at 37 degrees C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. The data show that rfxA must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in rfxA expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.

Metabolic and developmental effects resulting from deletion of the citA gene encoding citrate synthase in Aspergillus nidulans.

Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsADelta citADelta double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

AoxA is a major peroxisomal long chain fatty acyl-CoA oxidase required for beta-oxidation in A. nidulans.

Filamentous fungi can use a variety of fatty acids (FA) as sole carbon and energy sources. Aspergillus nidulans has been shown to possess both peroxisomal and mitochondrial beta-oxidation pathways. In these studies, the major peroxisomal long chain fatty acyl coenzyme A oxidase AoxA was identified. AoxA was shown to be localised to peroxisomes and deletion of the aoxA gene leads to reduced growth on long chain FA, but not on short chain FA. AoxA is predicted to be part of the same peroxisomal beta-oxidation pathway as the bifunctional protein FoxA. In addition, an aoxA(p)lacZ reporter gene construct is induced by short and long chain FA and the induction is dependent on the transcriptional regulators FarA, FarB and ScfA with FarA being required for the induction by short chain as well as long chain FA and FarB and ScfA being required for induction of aoxA by short chain FA. It is proposed that there are additional peroxisomal beta-oxidation pathways in A. nidulans, which include fatty acyl-CoA dehydrogenases with a partially overlapping substrate range and include a pathway for short chain FA.

Deletion and overexpression of the Aspergillus nidulans GATA factor AreB reveals unexpected pleiotropy.

The Aspergillus nidulans transcription factor AreA is a key regulator of nitrogen metabolic gene expression. AreA contains a C-terminal GATA zinc finger DNA-binding domain and activates expression of genes necessary for nitrogen acquisition. Previous studies identified AreB as a potential negative regulator of nitrogen catabolism showing similarity with Penicillium chrysogenum NreB and Neurospora crassa ASD4. The areB gene encodes multiple products containing an N-terminal GATA zinc finger and a leucine zipper motif. We deleted the areB gene and now show that AreB negatively regulates AreA-dependent nitrogen catabolic gene expression under nitrogen-limiting or nitrogen-starvation conditions. AreB also acts pleiotropically, with functions in growth, conidial germination and asexual development, though not in sexual development. AreB overexpression results in severe growth inhibition, aberrant cell morphology and reduced AreA-dependent gene expression. Deletion of either the DNA-binding domain or the leucine zipper domain results in loss of both nitrogen and developmental phenotypes.

Anomalously slow rates of reduction of iron(III) polypyridine complexes by triphenyl verdazyl radicals in acetonitrile.

The kinetics and mechanisms of the electron transfer reactions of [Fe(N-N)(3)](ClO(4))(3) [N-N = 2,2'-bipyridine, 1,10-phenanthroline] and [Fe(terpy)(2)](ClO(4))(3) [terpy = 2,2':6,2''-terpyridine] with a range of triphenyl verdazyl radicals have been investigated in acetonitrile at 25 degrees C and I = 0.05 mol dm(-3) (C(4)H(9))(4)NPF(6) using stopped-flow spectrophotometry. It was found that all three iron(III) complexes underwent extensive dissociation in acetonitrile and it was necessary to carry out the reactions in the presence of an excess of the relevant ligand. Under these conditions, reaction of the oxidant with the verdazyl radical resulted in an absorbance change that could be described by a single exponential. This was confirmed by global analysis of time-dependent spectra. A mechanism is proposed that accounts for the kinetic data. The rate constants for the electron transfer reaction are many orders of magnitudes less than would be predicted on the basis of the redox potentials and the self exchange rate constants of the reacting species.