Candida albicans SOD5 represents the prototype of anunprecedented class of Cu-only superoxide dismutasesrequired for pathogen defenseJulie E. Gleasona,1, Ahmad Galaleldeenb,1, Ryan L. Petersona, Alexander B. Taylorc, Stephen P. Hollowayc,Jessica Waninger-Saronib, Brendan P. Cormackd, Diane E. Cabellie, P. John Hartc,f,2, and Valeria Cizewski Culottaa,2

aDepartment of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; bDepartment ofBiological Sciences, St. Mary’s University, San Antonio, TX 78228; cDepartment of Biochemistry, University of Texas Health Science Center, San Antonio,TX 78229; dDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; eChemistry Department,Brookhaven National Laboratories, Upton, NY 11973-5000; and fDepartment of Veterans Affairs, Geriatric Research, Education, and Clinical Center, SouthTexas Veterans Health Care System, San Antonio, TX 78229

Edited by Sabeeha S. Merchant, University of California, Los Angeles, CA, and approved March 5, 2014 (received for review January 3, 2014)

The human fungal pathogens Candida albicans and Histoplasmacapsulatum have been reported to protect against the oxidativeburst of host innate immune cells using a family of extracellularproteins with similarity to Cu/Zn superoxide dismutase 1 (SOD1).We report here that these molecules are widespread throughoutfungi and deviate from canonical SOD1 at the primary, tertiary,and quaternary levels. The structure of C. albicans SOD5 revealsthat although the β-barrel of Cu/Zn SODs is largely preserved,SOD5 is a monomeric copper protein that lacks a zinc-binding siteand is missing the electrostatic loop element proposed to promotecatalysis through superoxide guidance. Without an electrostaticloop, the copper site of SOD5 is not recessed and is readily acces-sible to bulk solvent. Despite these structural deviations, SOD5 hasthe capacity to disproportionate superoxide with kinetics that ap-proach diffusion limits, similar to those of canonical SOD1. In cul-tures of C. albicans, SOD5 is secreted in a disulfide-oxidized formand apo-pools of secreted SOD5 can readily capture extracellularcopper for rapid induction of enzyme activity. We suggest that theunusual attributes of SOD5-like fungal proteins, including the ab-sence of zinc and an open active site that readily captures extra-cellular copper, make these SODs well suited to meet challenges inzinc and copper availability at the host–pathogen interface.

Eukaryotes are known to express two highly related classesof copper-containing superoxide dismutase (SOD) enzymes

that play widespread roles in oxidative stress resistance andsignaling. These two classes include an intracellular, largely cy-tosolic SOD1 (1) and an extracellular SOD (EC-SOD) (2), bothof which are bimetallic enzymes with copper and zinc cofactors.The redox active copper catalyzes the disproportionation of su-peroxide anion to oxygen and hydrogen peroxide, whereas thezinc helps stabilize the protein (3–5) and promotes pH in-dependence of the reaction (6–8). Additionally, all eukaryoticcopper and zinc SODs contain an active site channel thatconsists of a cluster of charges in loop VII that culminate with aninvariant arginine adjacent to the copper site. This element, com-monly known as the electrostatic loop, is proposed to providelong- and short-range guidance for the superoxide substrate,thereby facilitating the remarkable kinetics of the SOD reaction(2, 9, 10). SOD1 is among the fastest enzymes known, withrates (109 M−1 s−1) that approach diffusion limits (9, 11, 12).Very recently, certain pathogenic fungi have been reported to

express a class of extracellular proteins that are homologous toSOD1 and are covalently attached to the cell wall through GPIanchors. These SODs were first described for the opportunisticfungal pathogen Candida albicans. C. albicans is a commoncommensal microbe of the human gut, but under conditions ofa weakened immune system, the organism can become invasiveand pathogenic, with infections ranging from mild mucosalcandidiasis to life-threatening systemic disease (13). As withmany pathogens, C. albicans must evade the oxidative burst of

host immunity, and the intracellular SOD1 of C. albicans hasbeen shown to be important for virulence (14). C. albicans alsoharbors three genes for expressing SOD1-related GPI-anchoredproteins, namely, SOD4, SOD5, and SOD6. Of these genes, SOD5was shown to be critical for combating the oxidative burst of in-fection (15–17). C. albicans mutants lacking SOD5 are more sus-ceptible to killing by macrophages and neutrophils, and areassociated with increased reactive oxygen species production bymacrophages and neutrophils (16, 18, 19). Most notably, sod5Δ/Δmutant fungi exhibit decreased virulence in a mouse model forinfection (15). A similar GPI-anchored protein known as SOD3 wasreported for the respiratory human fungal pathogen Histoplasmacapsulatum (20). As with C. albicans SOD5, SOD3 helps protectH. capsulatum from killing by macrophages and is important forvirulence in a mouse model of infection (20). Despite the impor-tance of these EC-SOD1–like proteins in fungal pathogenesis, vir-tually nothing is known regarding their mechanism of action.In this study, we investigated the nature of these molecules by

focusing on C. albicans SOD5. The 3D structure of SOD5 wassolved by single-crystal X-ray diffraction, and the reactivity ofSOD5 with superoxide was examined in vitro and in C. albicanscultures in vivo. We report here that SOD5 represents an

Significance

Candida albicans is the most prevalent human fungal patho-gen. To combat the host immune response, C. albicans expressessuperoxide dismutase 5 (SOD5), a cell wall protein related toCu/Zn SODs. We find that SOD5 structure markedly deviatesfrom Cu/Zn SOD molecules. It is a monomeric copper-only SODthat lacks a zinc site and electrostatic loop. In spite of theseanomalies, SOD5 disproportionates superoxide at remarkablyrapid rates. When expressed in C. albicans, SOD5 can accumu-late outside the cell in an inactive form that can subsequently becharged for activity by extracellular copper. SOD5-like mole-cules are present in many fungal pathogens and appear to bespecialized for the metal and oxidative challenges presentedby the host immune system.

Author contributions: J.E.G., A.G., R.L.P., B.P.C., D.E.C., P.J.H., and V.C.C. designed re-search; J.E.G., A.G., R.L.P., S.P.H., and J.W.-S. performed research; J.E.G. and A.B.T. ana-lyzed data; and J.E.G., A.G., R.L.P., D.E.C., P.J.H., and V.C.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Coordinates and diffraction data for SOD5 structures coming from crys-tal forms A and B have been deposited in the Protein Data Bank, www.pdb.org [PDB IDcodes 4N3T (CuI) SOD5 and 4N3U (CuII) SOD5].1J.E.G. and A.G. contributed equally to this work.2To whom correspondence may be addressed. E-mail: pjhart@biochem.uthscsa.edu orvculotta@jhsph.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400137111/-/DCSupplemental.

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unprecedented class of Cu-only monomeric SODs that func-tion without a zinc cofactor and an electrostatic loop. Whensecreted from C. albicans, SOD5 is rapidly charged with ex-tracellular copper and the enzyme has the capacity to removesuperoxide at diffusion-limited rates. SOD5-like proteins ap-pear widely spread throughout pathogenic fungi, poised forthe unique challenges at the host–pathogen interface.

ResultsSOD5-Like Proteins Do Not Conform to Traditional Cu/Zn SOD Enzymes.Upon inspection of the primary sequences, we noted strikingdeviations between the fungal EC-SODs and canonical SOD1,an example of which is shown with C. albicans SOD5 in Fig. 1A.Although SOD5 possesses the copper binding histidines (H75,H77, H93, and H153), the disulfide cysteines (C87 and C162), andthe active arginine (R159) of SOD1, it is missing two of four zincbinding histidines as well as 17 residues of the all-importantelectrostatic loop (Fig. 1A). These aberrations are not unique toSOD5 but are also seen with C. albicans SOD4 and SOD6 andwithH. capsulatum SOD3 (Fig. 1B). Moreover, upon inspection ofavailable databases, SOD5-like proteins are predicted to occurthroughout Ascomycota and Basidiomycota fungi, each retainingthe SOD1-like copper site and disulfide but missing ligands forzinc and the electrostatic loop region (examples are provided inFig. 1B and Table S1).

Three-Dimensional Structure of C. albicans SOD5. To explore thestructure and mechanistic action of these curious fungal mole-cules, we used C. albicans SOD5 as a model. Recombinant SOD5lacking the N- and C-terminal signals for protein secretion andGPI anchorage, respectively, was expressed in Escherichia coli andreconstituted with copper to an occupancy of ∼80%. Extensiveattempts to reconstitute similarly with zinc or iron were un-successful, which was an unexpected finding, given the avidity of

zinc for the copper site in SOD1 (21) (Discussion). We determinedand refined the crystal structures of Cu(I)SOD5 and Cu(II)SOD5to resolutions of 1.4 and 1.75 Å, respectively. The 1.4-Å structure(crystal A) was determined using short-wavelength, high-fluxsynchrotron radiation, whereas the 1.75-Å structure (crystal B)was determined using longer wavelength, in-house X-rays in anexperiment designed to minimize photoreduction by the X-raybeam. Table S2 summarizes the diffraction data and proteinstructure refinement statistics.The overall architecture of SOD5 in the same orientation as

Saccharomyces cerevisiae SOD1 is shown in Fig. 2. Superimpo-sition of the two molecules (Fig. 2C) reveals that the key β-barreland zinc loop IV elements of SOD1 are largely retained inSOD5. The two molecules superimpose with an rmsd of 0.9 Å fortarget backbone atoms. SOD5 cysteines C87 and C162 form adisulfide that spatially coincides with that of SOD1 (Fig. 2). Asnotable distinctions, SOD5 contains an additional nine residuesat the C terminus for GPI anchorage and an expanded disulfidesubloop (Fig. S1), and it lacks zinc. With no electrostatic loop,the copper site in SOD5 appears more open than that of SOD1(Fig. 2). In addition, there is no evidence for a dimer interface inthe crystal structure or for self-association in the analytical ul-tracentrifugation sedimentation velocity profile (Fig. S2). Unlikedimeric and tetrameric SOD1 and EC-SOD, SOD5 is a mono-mer. Molecular modeling strongly suggests that the expansion ofthe disulfide loop of SOD5 precludes dimer formation throughsteric considerations (Fig. 2C and Fig. S1).The architecture and coordination geometries of Cu(I) and

Cu(II) bound to SOD5 are similar to those observed for S. cerevisiaeSOD1 (22, 23). In Cu(I) SOD5, the copper is coordinated byresidues H75, H77, and H153 in a pseudotrigonal planar ar-rangement and there is an absence of connecting electrondensity between the copper and the epsilon nitrogen of H93located ∼3.5 Å away (∼3.2 Å in SOD1). These observations areconsistent with protonation of the H93 side chain during thereduction of Cu(II) to Cu(I) (22, 23) (Fig. 3 A and B). Con-versely, in Cu(II) SOD5, this distance shortens to 2.8 Å (2.7 Åin yeast SOD1) and clear connecting electron density is ob-served between the copper ion and the epsilon nitrogen atomof H93 (Fig. 3C). These findings are consistent with deprotonationof H93, permitting the epsilon nitrogen of H93 to act as a ligand toCu(II) (22, 23) (Fig. 3D). In the absence of spectroscopic datataken from a crystal, however, care should be taken when corre-lating redox state with geometries. For example, in contrast to thetrigonal planar Cu(I) coordination geometries observed in SOD5and yeast SOD1, crystallographic determination of bovine SOD1,in which the copper had been forcefully reduced with exogenousreducing agents, revealed a four-coordinate copper with an intactimidazolate bridge. This finding was attributed to the influence ofcrystal packing forces and the SOD rack on copper oxidation state(24). Overall, however, the (His)3 coordinate copper observed incrystal A and the (His)4 coordinate copper in observed crystal Bare as expected for a copper-containing SOD enzyme during thetwo half-reactions of superoxide disproportionation.A remnant of the electrostatic loop in SOD5 is R159. In

SOD1, the equivalent R143 helps attract superoxide to thecopper site (12) and is anchored via carbonyl oxygens from G61and C57. These same interactions are preserved in SOD5, andSOD5 R159 is additionally stabilized by H-bonding with T90(Figs. 2A and 3 A and C). This positioning of R159 near thecopper site, together with the geometry of bound Cu(I) andCu(II), is highly reminiscent of the active site of Cu/Zn SODmolecules, despite no zinc and no electrostatic loop.

Rapid Kinetics of SOD5 Activity. Thus far, the only evidence sup-porting a SOD function for SOD5 has emerged from macro-phage and neutrophil infection studies, where the oxidative burstwas dampened by C. albicans expressing SOD5 (18, 19). Tomeasure SOD activity directly, we used pulse radiolysis, the de-finitive method for obtaining rate constants in SOD enzymes.We observed that SOD5 rapidly reacts with superoxide and that

Fig. 1. Sequence of SOD5 and SOD5-like proteins. (A) Alignment of theC. albicans (Ca) SOD5 against C. albicans and S. cerevisiae (Sc) SOD1. Copperligands, blue; zinc ligands, green; His bridges Zn(II) and Cu(II) in SOD1, blueand green boxed; disulfide Cys, purple; active site Arg, cyan; N- andC-terminal secretion and GPI anchorage extensions, yellow. Blue and blackarrows demark recombinant SOD5 from C. albicans and E. coli. A SOD1 elec-trostatic loop is underlined and a single dagger (†) marks the invariant Asp incopper-containing SODs. (B) SOD5 residues F72–N164 aligned against similardomains from select fungi. ΔEL, missing electrostatic loop; Hc, Ascomycotahuman pathogen H. capsulatum SOD3 (20); Pg, Basidiomycota plant pathogenPuccinia graminis XP_00336004; Pt, Ascomycota plant pathogen Pyrenophorateres XP_003305710.

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reactivity is proportional to enzyme concentration (Fig. 4A). Thekcat calculated from the linear regression of these data was foundto be 1.1 × 109 M−1 s−1 at pH 7.25 (Fig. 4B), a rate that closelyapproximates that determined per metal site in SOD1 (9). Thus,Cu-only SOD5 is a bona fide SOD enzyme with rates that ap-proach diffusion limits.The zinc cofactor in SOD1 promotes pH independence of kcat

from pH 6–9.5 (6–8). Although lacking zinc, SOD5 is relativelyunaffected up to pH 8.0, with maximal activity at pH 6.0, wherekcat = 1.8 × 109 M−1 s−1 (Fig. 4C). The precipitous drop in activityat pH ≥ 8.5 was readily reversible upon lowering the pH, even inthe presence of metal chelators and under conditions when SOD5was pulsed with superoxide and generated micromolar H2O2(Fig. 4C). Hence, there is minimal peroxide damage to the enzymeat high pH, and the copper cofactor is retained. It is noteworthythat in the human host, C. albicans is not expected to experiencepH conditions that exceed 8.0 and, moreover, the organism iscapable of neutralizing the pH of its environment (25).SOD1 activity decreases under conditions of high ionic strength,

presumably due to ionic shielding of the steering forces in theelectrostatic loop (9, 10, 12). Surprisingly, SOD5 also exhibiteda decrease in kcat at increasing ionic strength with slopes of −0.6and −0.35 at pH 6.0 and pH 7.25, respectively (Fig. 4D). Thus,even without an electrostatic loop, steering forces may participatein guiding substrate to the SOD5 active site (Discussion).

SOD5 Functions as a Copper-Dependent SOD in C. albicans. Althoughrecombinant SOD5 expressed in E. coli is clearly an efficientSOD, it was critical to evaluate SOD5 expressed from its native

host, where the protein is processed through the secretorypathway. SOD5 is normally anchored in the cell wall, but theharsh treatments needed to extract SOD5 protein from the cellwall (17) would prohibit recovery of active enzyme. Also, theabundant cell surface reductases in C. albicans interfere withSOD assays in intact cells (26). To circumvent these issues, weexpressed SOD5 lacking the GPI anchor, such that secretedSOD5 could be recovered in the growth medium, precisely aswas done with Histoplasma SOD3 (20). Recombinant SOD5-170missing the C-terminal GPI anchor signal was secreted fromC. albicans in a heavily glycosylated form, as indicated by strongreactivity with endoglycosidases (Fig. 5A). By a native gel assayfor SOD enzymes, SOD5-170 activity was strongly induced bysupplementing cultures with copper (Fig. 5B) but not zinc (Fig.5C), and the same was true for a larger version of SOD5, namely,SOD5-204, which more closely approximates full-length SOD5anchored to the cell wall at position 205 (Fig. S3B). We alsoexamined the disulfide status of SOD5 using a redox Westernblot (27). In the case of fungal SOD1, disulfide oxidation istypically coupled with copper insertion (27, 28). However, withSOD5, the disulfide appears oxidized under all conditions, evenwithout the copper supplements needed to activate the enzymemaximally (Fig. 5D). Disulfide oxidation in SOD5 does not ap-pear to be dependent on copper.Eukaryotic Cu/Zn SODs are regulated posttranslationally

through the controlled insertion of copper and oxidation of thedisulfide bond. With intracellular SOD1, both modifications areaccomplished through the copper chaperone for SOD1 (CCS)(28–30), whereas EC-SOD matures inside the secretory pathwayusing protein disulfide isomerases in the endoplasmic reticulumto oxidize disulfides (31) and copper derived from ATP7a, aGolgi copper-transporting ATPase (32). We expected matura-tion of C. albicans SOD5 to rely likewise on secretory pathwaycopper and the fungal ATP7a. As predicted, SOD5 activity didnot require the cytosolic CCS copper chaperone because activitywas not affected in a ccs1Δ/Δ deletion mutant (33) (Fig. 6A). Sur-prisingly, however, there were no deleterious effects of mutating

Fig. 2. Overall structure of SOD5 and comparison with SOD1. The 3Dstructure of C. albicans SOD5 is compared with S. cerevisiae SOD1, whereyellow, blue, and green spheres correspond to disulfide bonds, Cu atoms,and Zn atoms, respectively. (A) C. albicans SOD5. The β-barrel is blue, loop IVis green, loop VII is yellow, and His93 is orange. H-bonds and Cu–ligandinteractions are yellow dashes. (B) S. cerevisiae SOD1. The β-barrel is pink,loop IV is blue, loop VII is red, and His63 is orange. H-bonds and metal–ligandinteractions are yellow dashes. (C) Superimposition of SOD5 and SOD1. Theboxed region of the disulfide loop (a subelement of loop IV) preventshomodimerization of SOD5 (Fig. S1).

Fig. 3. Copper coordination in SOD5. (A) Sigma-A (s-A) 1.4-Å weightedelectron density with coefficients 2mFo-DFc contoured at 1.2 s on the Cu(I) SOD5 structure in divergent stereo. The color coding is as in Fig. 2,except His93 is tan and a Tris molecule is pink. In lieu of Zn, a hydrogenbond is observed between the nonliganding imidazole nitrogen of H93and E110. Select H-bonds and Cu(I)–ligand interactions are orange andblue dashes, respectively. (B) Cu(I) pseudotrigonal planar geometry(yellow dashes) with axial ligands (red dashes). The H93 imidazole ni-trogen is ∼23° off-axis. (C ) s-A 1.75 Å weighted electron density for Cu(II)SOD5 as calculated as in A. Note connecting electron density between Cu(II) and H93. (D) Cu(II) coordination by four His ligands (blue dashes) withlonger pseudoaxial interactions (red dashes).

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CCC2 encoding the ATP7a for secretory pathway copper (34).The same was true whether we tested SOD5-170 that had ac-cumulated over 16 h in culture (Fig. 6B) or SOD5-170 that wasfreshly secreted over 1 h (Fig. 6C). Mutations in ccc2 also did notinhibit oxidation of the SOD5 disulfide (Fig. 6D). Disulfide ox-idation may occur in the secretory pathway, as is the case withmammalian EC-SOD (31). However, unlike EC-SOD, SOD5does not require the Golgi ATP7a for copper. If copper loadingoccurs in the secretory pathway, there must be a non-ATP7asource for the metal; alternatively, the enzyme may be chargedwith copper outside the cell. To study SOD5 activation by ex-tracellular copper, cells were removed from 16-h cultures, wherea large fraction of the accumulated SOD5-170 is inactive butdisulfide-oxidized (as in Fig. 5 B and D). In this cell-free milieu,SOD5-170 activity was rapidly induced by copper (Fig. 6E) andthe same results were obtained with full length SOD5-204 (Fig.S3C). Thus, SOD5 can accumulate outside the cell as an apo-disulfide–oxidized protein that is primed for rapid activation byextracellular copper. The capture of extracellular copper bySOD5-like molecules may be particularly relevant during the“copper burst” of the host immune response (35) (Discussion).

DiscussionFungi such as C. albicans express a previously unidentified classof copper-only SOD enzymes. To our knowledge, these are thefirst copper-containing SODs from any organism (prokaryotic oreukaryotic) reported to lack the extended loop VII, which con-stitutes the electrostatic loop in eukaryotic SOD1 and EC-SOD.Despite this anomaly, our studies of C. albicans SOD5 showreactivity with superoxide at rates that approach diffusionlimits, similar to SOD1. The fungal enzyme also exhibits spe-cial metal cofactor attributes, including an open copper siteand no zinc site, which are structural signatures of these fungalenzymes that could represent prime targets for future drug de-sign strategies.

Based on our analysis of C. albicans SOD5, the absence of anextended electrostatic loop results in a copper site that does notlie at the bottom of a tapering funnel as in SOD1. Instead, thecopper site is more open, which might promote reactivity withsubstrates other than superoxide or could enhance capture ofcopper from the extracellular environment. We show here thatSOD5 can accumulate outside the yeast cell in a disulfide-oxidizedapo-state, primed for rapid activation by extracellular copper.With an open copper site, the secreted enzyme appears to acquirecopper on its own, independent of an accessory copper chaperone.In its natural setting, C. albicans SOD5 relies on the animal hostfor copper, and one intriguing source is the macrophage. Duringthe innate immune response, macrophages release copper in anattempt to kill invading microbes through copper toxicity. Thelevels of copper in the macrophage phagolysosome can be ex-traordinarily high (100–300 μM), sufficient to induce copper stressresponses in invading microbes and inhibit invasive growth ofcopper-sensitive C. albicansmutants (35–40). Based on our studiesof secreted SOD5, this high level of host copper should be fa-vorable for SOD5 activation. In essence, SOD5 could “turn thesword” on the copper attack, exploiting host copper to transformitself into an antioxidant defense for the pathogen. This concept ofusing host copper for oxidative stress defense has also been pro-posed for uropathogenic E. coli that secretes a copper bindingsiderophore (yersiniabactin) with SOD mimetic activity (41).C. albicans SOD5 demonstrates an inability to bind zinc. Not

only is a zinc site missing, but zinc does not readily bind the coppersite. In SOD1, zinc can occupy either the copper or zinc site (21).Upon close inspection of the SOD5 copper site, a backbone car-bonyl oxygen (from V151) that is absent in SOD1 sits 4 Å axially

Fig. 4. Pulse radiolysis measurements of SOD5 reactivity (A) Time-dependentdisappearance of superoxide at pH 7.25 as a function of SOD5 concentra-tion: black (7.7 μM), light blue (3.8 μM), red (1.9 μM), green (0.97 μM), blue(0.48 μM), pink (0.24 μM). (B) Linear regression of the data from A was usedto calculate pseudo–first-order rate constants. (C) Calculated second-orderrate constants as a function of pH. Blue and black stars, SOD5 preincubatedat pH 9.5 and pulsed with superoxide at pH 10.0 before analysis of activityat pH 7.3. (D) Experimentally determined rate constants for the loss of su-peroxide as a function of varying ionic strength (NaCl) at pH 6.0 (red) and pH7.25 (blue). A slope of −0.6 (pH 6.0) and −0.35 (pH 7.25) is observed.

Fig. 5. Copper activation of SOD5 expressed in C. albicans. Growth mediumfrom 16 h C. albicans cultures expressing SOD5-170 or transformed with emptyvector (EV) (B) was subjected to PNGase endoglycosidase treatment beforeimmunoblot analysis for SOD5 [A, C (Lower), and D] or SOD activity by thenative gel assay using samples not treated with PNGase [B and C (Upper)]. (A)Collapse of SOD5 upon PNGase treatment (+) indicates extensive N-linked gly-cosylation. − indicates untreated samples. C. albicans transformed with emptyvector or expressing SOD5-170 was treated with 10 μM CuSO4 for the indicatedtimes (B) or with the indicated concentrations of CuSO4 or ZnCl2 for 1 h (C). (D)Redox Western blot, where upward shift in mobility with DTT (+) indicatesoxidized disulfide (27). Red and Ox, positions of disulfide-reduced and disulfide-oxidized SOD5, respectively. Lanes 1–2, recombinant disulfide-oxidizedSOD5 from E. coli as a control; lanes 3–5, SOD5-170 from C. albicans cultures,where +Cu indicates cells treated with 10 μM CuSO4 for 1 h before SOD5-170 analysis.

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from the pseudotrigonal plane formed by the Cu binding ligandsH75, H77, and H153 (Fig. 3B). Such a trigonal planar copper sitewith an outer-sphere axial oxygen atom may be incompatible withthe tetrahedral and octahedral coordination preferences of zinc.During infection, zinc levels can greatly fluctuate, because the hosteither starves pathogens of zinc or attacks the microbe with toxicdoses of the metal (42, 43). Because SOD5 neither requires zincnor allows zinc to misincorporate into the copper site, this SOD isresistant to such extremes in zinc. It is worth noting that thepathogenic Mycobacterium tuberculosis expresses a copper-con-taining SodC that has retained the extended loop VII (equivalentto the electrostatic loop) but, like SOD5, is missing ligands forzinc binding (44). Remarkably, the same two zinc ligands aremissing in bacterial SodC and fungal SOD5, yet both retain theAsp that would represent the fourth zinc ligand in Cu/Zn SODs(44) (Fig. 1B). In the absence of binding zinc, this Asp H-bonds tocopper binding H90 in SodC, and to copper binding H75 in SOD5(Fig. S4). It is remarkable that nature has preserved this Asp toconnect to the copper site but through different routes in Cu/Znvs. Cu-only SODs (Fig. S4).Despite lacking the extended electrostatic loop, we observed

an effect of ionic strength on kinetics of SOD5 reactivity withsuperoxide, indicative of an electrostatic guidance system forsubstrate. The remnant of the electrostatic loop in SOD5includes the active site R159 that, together with the Cu(I) or Cu(II)ion, may account for the effects observed. It is also possible thatthe fungal family of Cu-only SODs evolved with an alternativeelectrostatic guidance system. In fact, an auxiliary electrostaticguidance system was previously proposed for SOD1, where thereaction rate of Xenopus laevis SOD1 was seen to increase ratherthan decrease when all of the electrostatic loop charges were

neutralized by mutagenesis (45). In addition to substrate guidance,the extended electrostatic loop of SOD1 helps stabilize the zincsite, and the reverse is true: Zinc helps position the electrostaticloop (3, 4, 21). Because the fungal Cu-only SODs lack both fea-tures, it is possible that in Cu/Zn SODs, the electrostatic loopcoevolved with zinc binding properties, features built onto a pri-mordial SOD5-like molecule.

Materials and MethodsRecombinant SOD5 from E. coli. Recombinant SOD5 spanning sequences 27–181was expressed in E. coli and purified as described in SI Materials andMethods. Toreconstitute with metals, the SOD5 sample was dialyzed against 50 mM sodiumacetate (pH 5.5) and 0.25 mM CuSO4, followed by dialysis against the samebuffer without copper to remove adventitiously bound metal ions. The recon-stituted protein was subsequently dialyzed against 25 mM Tris (pH 8.0) andconcentrated to 3.2 mg/mL for use in pulse radiolysis experiments (as describedin SI Materials and Methods) and to 20 mg/mL in preparation for analyticalultracentrifugation experiments (as described in SI Materials and Methods) andcrystallization trials. This protocol was repeated using ZnCl2 and FeCl3 instead ofCuSO4. All three SOD5 samples were used in crystallization trials.

Structure Determination. Purified SOD5 proteins that had been dialyzedagainst CuSO4, ZnCl2, and FeCl3-containing buffer solutions were concen-trated to 20 mg/mL and set up in crystallization experiments using an ArtRobbins Instruments Phoenix crystallization robot and commercially avail-able crystallization kits in the X-ray Crystallography Core Laboratory atUniversity of Texas Health Science Center at San Antonio. Initial hits wereoptimized at 20 °C using the hanging drop vapor diffusion method. Iso-morphous crystals were grown from two different crystallization conditions.These isomorphous crystals are designated as crystal forms A and B, re-spectively, to reflect the differences in reservoir solutions. For crystal form A,SOD5 was mixed with an equal volume of reservoir solution containing2.4 M ammonium sulfate and 0.1 M bicine (pH 9.0). For crystal form B, thereservoir solution was 1.6 M sodium citrate (pH 6.5) and 0.1 M Hepes (pH7.5). Prism-shaped crystals appeared within 3 d in both conditions. Suitablespecimens were extracted with nylon loops, and the excess liquid wascarefully wicked away before flash-cooling in liquid nitrogen. Initial datasetswere collected in-house, and the structures were determined by molecularreplacement as implemented in the program PHASER (46), using the humanG37R variant of SOD1 [Protein Data Bank (PDB) ID code 1AZV] as the searchmodel. The structures were refined with the PHENIX suite of programs (47),and manual model adjustments were performed after each round of re-finement in the program Coot (48). Crystals grown from SOD5 protein thathad been dialyzed against ZnCl2 and FeCl3 were completely devoid of metalions and were not studied further. Copper-containing crystal forms A and Bwere subsequently taken to beamline 24-ID-E at the Advanced PhotonSource, Argonne National Laboratory, to obtain the highest resolutionpossible. X-ray fluorescence scans on the crystals revealed a strong signal forcopper with the expected absorption edge at 1.38 Å. Diffraction data wereindexed and scaled using the X-ray Detector Software (XDS) program. Thecoordinates of the SOD5 protein structures determined in-house were re-fined against the high-resolution data from the synchrotron as describedabove. The copper ion coordination was pseudotrigonal planar, consistentwith the presence of Cu(I) in both crystal forms, possibly as a result of thephotoreduction that often occurs using synchrotron radiation of shortwavelength and very high flux. The highest resolution dataset at a resolu-tion of 1.4 Å came from a form A crystal. In an effort to avoid photore-duction, 120° of diffraction data from a fresh form B crystal were measuredin-house at a resolution of 1.75 Å using oscillation images at 1° with anexposure time of 1 min (one oscillation image per degree per minute).Connecting density from the copper to His93 is present in this formB structure, indicating it contains a significant fraction of four-coordinateCu(II). All structural figures were created using the PyMol (Molecular GraphicsSystem, version 1.5.0.4; Schrödinger, LLC) program. Coordinates and diffrac-tion data for SOD5 structures coming from crystal forms A and B have beendeposited into the PDB with ID codes 4NT3 and 4NTU, respectively.

Biochemical Analyses of SOD5 Secreted from C. albicans. The growth mediumfrom C. albicans strains secreting SOD5-170 and SOD5-204 was concentratedas described in SI Materials and Methods. Samples for immunoblots weretreated with peptide-N-glycosidase f before denaturing gel electrophoresisand probing with an anti-SOD5 antibody. Samples for SOD activity by thenative gel assay (27) were assayed without prior PNGase treatment. Detailsare provided in SI Materials and Methods. Redox Western blots of SOD5

Fig. 6. Activation of SOD5 by extracellular copper. SOD5-170 secretedfrom WT or the indicated mutants of C. albicans was analyzed for SODactivity and protein levels (A–C and E) as in Fig. 5C and for disulfide oxida-tion (D) as in Fig. 5D. Cu (+), samples treated with 10 μM CuSO4 for 1 h (A–D)or for the indicated times (E). (A and B) SOD5 was secreted for 16 h beforecopper addition. The high level of SOD5 accumulated over this timeframe requires additional copper for maximal activity. (C and D) SOD5secreted for only 1 h, where the medium has ample copper to activatethis low level of enzyme maximally. Fourfold more sample was analyzed in Ccompared with B. (E) Cells were removed from 16-h cultures, and medium waseither reconstituted with cells (+ cells) or not reconstituted (− cells) beforecopper addition.

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were carried out using nonreducing gel electrophoresis as described (27).Concentrated medium samples were first treated with PNGase in the ab-sence of DTT, followed by inactivation of PNGase by incubation at 75 °C for10 min. Samples were then incubated at 70 °C for 10 min with SDS gel-loading buffer in the absence or presence 100 mM DTT before SDS gelelectrophoresis. Under these conditions, 0.5 μg of recombinant disulfide-oxidized SOD5 from E. coli exhibits a small upward shift in mobility uponreduction of the disulfide with DTT (Fig. 5D, lanes 1 and 2), as has beenshown for SOD1 (27). The same upward shift was seen with SOD5 secretedfrom C. albicans. Because the disulfide cysteines are the only cysteines inSOD5, this reactivity with DTT is presumed to reflect reduction of a preex-isting disulfide, as has been shown for SOD1.

ACKNOWLEDGMENTS. We thank D. Kornitzer for kind gifts of yeast strains,and B. Demeler and V. Schirf for assistance in analytical ultracentrifugationexperiments. This work was funded by National Institutes of Health GrantsR21 AI097715 (to V.C.C.), R37 GM50016 (to V.C.C.), and R01AI046223 (to B.P.C.)and by Veterans Administration Grant VA 1I01BX000506 and Robert A. WelchFoundation Grant AQ-1399 (to P.J.H.). J.E.G. and R.L.P. were supported byGrant T32 CA009110, and A.G. and J.W.-S. were supported by a St. Mary’sUniversity grant and the Biaggini Research Program. The work at BrookhavenNational Laboratory was carried out at the Accelerator Center for Energy Re-search, supported by the US Department of Energy Office of Science, Divisionof Chemical Sciences, Geosciences, and Biosciences under Contract DE-AC02-98CH10886. The Center for Analytical Ultracentrifugation of MacromolecularAssemblies is supported by the Office of the Vice President for Research at theUniversity of Texas Health Science Center.

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