FFAME.org :: Thomas Lyons's Publications

Directed evolution of a filamentous fungus for thermotolerance
de Crecy, E Jaronski, S Lyons, B Lyons, TJ Keyhani, NO
BMC Biotechnology 9 74 (2009)
<Abstract>

Background: Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications. Consequently, there is tremendous interest in methodology that can use the power of genetics to develop strains with improved performance. For example, Metarhizium anisopliae is a broad host range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. However, it use is limited by the relatively low tolerance of this species to abiotic stresses such as heat, with most strains displaying little to no growth between 35-37 degrees C. In this study, we used a newly developed automated continuous culture method called the Evolugator(TM) which takes advantage of a natural selection-adaptation strategy, to select for thermotolerant variants of M. anisopliae strain 2575 displaying robust growth at 37 degrees C. Results: Over a 4 month time course, 22 cycles of growth and dilution were used to select 2 thermotolerant variants of M. anisopliae. Both variants displayed robust growth at 36.5 degrees C, whereas only one was able to grow at 37 degrees C. Insect bioassays using Melanoplus sanguinipes (grasshoppers) were also performed to determine if thermotolerant variants of M. anisopliae retained entomopathogenicity. Assays confirmed that thermotolerant variants were, indeed, entomopathogenic, albeit with complex alterations in virulence parameters such as lethal dose responses (LD50) and median survival times (ST50). Conclusion: We report the experimental evolution of a filamentous fungus via the novel application of a powerful new continuous culture device. This is the first example of using continuous culture to select for complex phenotypes such as thermotolerance. Temperature adapted variants of the insect-pathogenic, filamentous fungus M. anisopliae were isolated and demonstrated to show vigorous growth at a temperature that is inhibitory for the parent strain. Insect virulence assays confirmed that pathogenicity can be retained during the selection process. In principle, this technology can be used to adapt filamentous fungi to virtually any environmental condition including abiotic stress and growth substrate utilization.

Antagonism of Human Adiponectin Receptors and Their Membrane Progesterone Receptor Paralogs by TNF alpha and a Ceramidase Inhibitor
Kupchak, BR Garitaonandia, I Villa, NY Smith, JL Lyons, TJ
Biochemistry 48 (24) 5504-5506 (2009)
<Abstract>

The progestin and AdipoQ receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are nonclassical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1(S),2(R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNF alpha, a homologue of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.

Adiponectin identified as an agonist for PAQR3/RKTG using a yeast-based assay system
Garitaonandia, I Smith, JL Kupchak, BR Lyons, TJ
J. Recept. Signal Transduction 29 (1) 67-73 (2009)
<Abstract>

The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered "orphan" receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.

Sphingolipids Function as Downstream Effectors of a Fungal PAQR
Villa, NY Kupchak, BR Garitaonandia, I Smith, JL Alonso, E Alford, C Cowart, LA Hannun, YA Lyons, TJ
Mol. Pharmacol. 75 (4) 866-875 (2009)
<Abstract>

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as D-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steady-state levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.

Dissecting the regulation of yeast genes by the osmotin receptor
Kupchak, BR Villa, NY Kulemina, LV Lyons, TJ
Biochem. Biophys. Res. Comm. 374 (2) 210-213 (2008)
<Abstract>

The Izh2p protein from Saccharomyces cerevisiae is a receptor for the plant antifungal protein, osmotin. Since Izh2p is conserved in fungi, understanding its biochemical function could inspire novel strategies for the prevention of fungal growth. However, it has been difficult to determine the exact role of Izh2p because it has pleiotropic effects on cellular biochemistry. Herein, we demonstrate that Izh2p negatively regulates functionally divergent genes through a CCCTC promoter motif. Moreover, we show that Izh2p-dependent promoters containing this motif are regulated by the Nrg1p/Nrg2p and Msn2p/Msn4p transcription factors. The fact that Izh2p can regulate gene expression through this widely dispersed element resents a reasonable explanation of its pleiotropy. The involvement of Nrg1p/Nrgp2 in Izh2p-dependent gene regulation also suggests a role for this receptor in regulating fungal differentiation in response to stimuli produced by plants. (C) 2008 Elsevier Inc. All rights reserved.

Heterologous expression of human mPR alpha, mPR beta and mPR gamma in yeast confirms their ability to function as membrane progesterone receptors
Smith, JL Kupchak, BR Garitaonandia, I Hoang, LK Maina, AS Regalla, LM Lyons, TJ
Steroids 73 (11) 1160-1173 (2008)
<Abstract>

The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPR alpha, mPR beta and mPR gamma, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPR alpha, mPR beta and mPR gamma can sense and respond to progesterone with EC50 values that are physiologically relevant. Agonist profiles also show that mPR alpha, mPR beta and mPR gamma are activated by ligands, such as 17 alpha-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs; have been renamed as mPR delta (PAQR6) and mPR epsilon (PAQR9). Additional characterization of mPR gamma and mPRa indicates that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs; as a novel class of G-protein coupled receptor. (c) 2008 Elsevier Inc. All rights reserved.

Probing the mechanism of FET3 repression by Izh2p overexpression
Kupchak, BR Garitaonandia, I Villa, NY Mullen, MB Weaver, MG Regalla, LA Kendall, EA Lyons, TJ
Biochim. Biophys. Acta 1773 (7) 1124-1132 (2007)
<Abstract>

We previously reported a role for the IZH2 gene product in metal ion metabolism. Subsequently, Izh2p was also identified as a member of the PAQR family of receptors and, more specifically, as the receptor for the plant protein osmotin. In this report, we investigate the effect of Izh2p on iron homeostasis. We show that overproduction of Izh2p prevents the iron-dependent induction of the Fet3p component of the high-affinity iron-uptake system and is deleterious for growth in iron-limited medium. We demonstrate that the effect of Izh2p requires cAMP-dependent kinase and AMP-dependent kinase and is not mediated by general inhibition of the Aft1p iron-responsive transcriptional activator. We also show that lzh2p-overproduction negatively regulates Nrg1p/Nrg2p- and Msn2p/Msn4p-dependent reporters. Furthermore, we show that the Nrg1p/Nrg2p and Msn2p/Msn4p pairs are epistatic to each other with respect to their effects on FET3 expression. Finally, we show that the mechanism by which PAQR receptors activate signal transduction pathways is likely to be conserved from yeast to humans. (C) 2007 Elsevier B.V. All rights reserved.

Transport and Storage of Metal Ions in Biology
Lyons, TJ Eide, DJ
Biological Inorganic Chemistry: Structure and Reactivity, ed. I. Bertini, H. Gray, E. Stiefel, and J.S. Valentine, University Science Books 57-77 (2007)

Zinc in yeast: mechanisms involved in homeostasis
Regalla, LM Lyons, TJ
Molecular Biology of Metal Homeostasis and Detoxification: From Microbes to Man, ed. Markus J. Tamás and Enrico Martinoia, Springer 37-58 (2006)

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function
Ellis, CD Wang, FD MacDiarmid, CW Clark, S Lyons, T Eide, DJ
J. Cell. Biol. 166 (3) 325-335 (2004)
<Abstract>

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.

Metalloregulation of yeast membrane steroid receptor homologs
Lyons, TJ Villa, NY Regalla, LM Kupchak, BR Vagstad, A Eide, DJ
Proc. Natl. Acad. Sci. USA 101 (15) 5506-5511 (2004)
<Abstract>

Zinc is an essential micronutrient that can also be toxic. An intricate mechanism exists in yeast that maintains cellular zinc within an optimal range. The centerpiece of this mechanism is the Zap1p protein, a transcription factor that senses zinc deficiency and responds by up-regulating genes involved in zinc metabolism. A microarray screen for novel Zap1p target genes suggested a role in zinc homeostasis for four homologous yeast genes. The expression of two of these genes, YDR492w and YOL002c, suggested direct regulation by Zap1p, whereas the expression of YOL002c and a third homologous gene, YOL101c, was induced by high zinc. YDR492w and YOL002c are confirmed to be direct Zap1p target genes. The induction of YOL002c and YOL101c by toxic metal ion exposure is shown to be mediated by the Mga2p hypoxia sensor. Furthermore, YOL101c is induced by deletion of the Aft1p iron-responsive transcription factor. These three genes, along with a fourth yeast homolog, YLR023c, have phenotypic effects on zinc tolerance and Zap1p activity. Because of their metalloregulation, zinc-related phenotypes, and highly conserved motifs containing potential metal-binding residues, this family has been renamed the IZH gene family (implicated in Zinc Homeostasis). Furthermore, these genes are regulated by exogenous fatty acids, suggesting a dual role in lipid metabolism. The IZH genes encode membrane proteins that belong to a ubiquitous protein family that includes hemolysin III and vertebrate membrane steroid receptors. We propose that the IZH genes affect zinc homeostasis either directly or indirectly by altering sterol metabolism.

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): A dimer-dimer model of LYS7-SOD association and copper delivery
Hall, LT Sanchez, RJ Holloway, SP Zhu, HN Stine, JE Lyons, TJ Demeler, B Schirf, V Hansen, JC Nersissian, AM Valentine, JS Hart, PJ
Biochemistry 39 (13) 3611-3623 (2000)
<Abstract>

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 Angstrom crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.

The copper transport protein Atox1 promotes neuronal survival
Kelner, GS Lee, MH Clark, ME Maciejewski, D McGrath, D Rabizadeh, S Lyons, T Bredesen, D Jenner, P Maki, RA
J. Biol. Chem. 275 (1) 580-584 (2000)
<Abstract>

Atox1, a copper transport protein, was recently identified as a copper dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.

The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis
Lyons, TJ Nersissian, A Huang, HJ Yeom, H Nishida, CR Graden, JA Gralla, EB Valentine, JS
J. Biol. Inorg. Chem. 5 (2) 189-203 (2000)
<Abstract>

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pK(a). The mutants H71C and D83A did not bind Co2+ in a wildtype-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast
Lyons, TJ Gasch, AP Gaither, LA Botstein, D Brown, PO Eide, DJ
Proc. Natl. Acad. Sci. USA 97 (14) 7957-7962 (2000)
<Abstract>

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Biological chemistry of copper-zinc superoxide dismutase and its link to amyotrophic lateral sclerosis
Lyons, TJ Gralla, EB Valentine, JS
Met. Ions Biol. Syst. 36 (1) 125-177 (1999)

The dark side of dioxygen biochemistry
Valentine, JS Wertz, DL Lyons, TJ Liou, LL Goto, JJ Gralla, EB
Curr. Op. Chem Biol. 2 (2) 253-262 (1998)
<Abstract>

The cellular biochemistry of dioxygen is Janus-faced. The good side includes numerous enzyme-catalyzed reactions of dioxygen that occur in respiration and normal metabolism, while the dark side encompasses deleterious reactions of species derived from dioxygen that lead to damage of cellular components. These reactive oxygen species have historically been perceived almost exclusively as agents of the dark side, but it has recently become clear that they play beneficial roles as well.

Metal ion reconstitution studies of yeast copper-zinc superoxide dismutase: the "phantom" subunit and the possible role of Lys7p
Lyons, TJ Nersissian, A Goto, JJ Zhu, H Gralla, EB Valentine, JS
J. Biol. Inorg. Chem. 3 (6) 650-662 (1998)
<Abstract>

Using a corrected molar extinction coefficient for yeast apo copper-zinc superoxide dismutase (CuZnSOD), we have confirmed that the metal binding properties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one Co2+ per protein dimer under the conditions in which the bovine and human CuZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties characteristic of the two Cu2+ plus two Co2+ per dimer or four Cu2+ per dimer metal-substituted bovine apo CuZnSOD derivatives were obtained for the yeast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu2+ plus one Co2+ per dimer or two Cu2+ per dimer. This half-metallated yeast CuZnSOD has been characterized by UV-visible and EPR spectroscopy as well as by native polyacrylamide gel electrophoresis. We conclude that yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be reconstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "phantom" subunit is not competent to bind metals in this fashion. Furthermore, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisiae that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn2+, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased propensity of the yeast protein to undergo changes that can occur in all CuZnSODs, and may have implications for amyotrophic lateral sclerosis.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein
Lyons, TJ Liu, HB Goto, JJ Nersissian, A Roe, JA Graden, JA Cafe, C Ellerby, LM Bredesen, DE Gralla, EB Valentine, JS
Proc. Natl. Acad. Sci. USA 93 (22) 12240-12244 (1996)
<Abstract>

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the mild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

OXYGENATION OF COBALT(II)-SUBSTITUTED LIMULUS-POLYPHEMUS HEMOCYANIN - KINETICS, CD, AND MCD STUDIES
Larrabee, JA Baumann, TF Chisdes, SJ Lyons, TJ
Inorg. Chem. 31 (17) 3630-3635 (1992)
<Abstract>

Cobalt(II)-substituted Limulus polyphemus (CoHcy) is characterized by circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. At neutral pH, the active site Co(II)'s are mostly aquoCoHcy, which gives rise to weak CD but intense low-temperature (4.2 K) MCD spectral features at 571, 552, and 526 nm. At higher pH's CoHcy is mostly hydroxoCoHcy and still has a weak visible CD spectrum, but three near-UV CD peaks at 372, 340, and 316 nm appear which are proposed to arise from ligand to metal charge transfer (LMCT). The 4.2 K MCD spectrum of hydroxoCoHcy is very rich with peaks at 304, 324, 355, 526, 556, 571, 616, and 642 nm. These spectra can be interpreted in terms of approximate C3, ligand symmetry about each active-site cobalt(II) with a (HiS)30 ligand set. In the case of aquoCoHcy, the 0 ligand comes from a coordinated water, whereas, in hydroxoCoHcy, the 0 ligand comes from hydroxide. The hydroxoCoHcy spectrum in the d-d transition region has too many peaks to be accounted for solely from spin-allowed transitions; therefore, it is proposed that the 616- and 642-nm bands arise from spin-forbidden 4A2 --> 2A2(G),2E(G) transitions which gain intensity from the nearby spin-allowed 4A2 --> 4E(P) and 4A2(P) transitions. The aquoCoHcy and hydroxoCoHcy MCD spectral features are strikingly similar to those of the low-pH and high-pH forms of cobalt-substituted carbonic anhydrases, respectively. HydroxyCoHcy rapidly oxygenates (k = 500 M-1 s-1) to form oxyCoHcy, which has strong CD bands at 332, 413, 518, and 618 nm. These bands are supportive of an oxyCoHcy active site, which contains mu-1,2-peroxo, mu-hydroxo dibridged Co(III) dimers. The 332- and 413-nm bands are due to pi(a)* --> d(sigma)* and pi(b)* --> d(sigma)* O2(2-) --> Co(III) LMCT, while the 518- and 618-nm CD bands are d-d transitions arising from six-coordinate Co(III).