Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Masha G. Savelieff*, Tiffany D. Wilson*, Youssef Elias†, Mark J. Nilges‡, Dewain K. Garner*, and Yi Lu*†§

Departments of *Chemistry and †Biochemistry and ‡Illinois EPR Research Center, University of Illinois, Urbana, IL 61801

Edited by Edward I. Solomon, Stanford University, Stanford, CA, and approved March 18, 2008 (received for review November 29, 2007) The cupredoxin fold is an important motif in numerous proteins the geometry and ligand donor set about the copper active site that are central to several critical cellular processes ranging from (12). For example, blue copper proteins, such as plastocyanin, aerobic and anaerobic respiration to catalysis and iron homeosta- azurin, laccase, NIR, and ceruloplasmin, have a mononuclear sis. Three types of copper sites have been found to date within copper active site, the copper–thiolate bond of which resides in cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, a trigonal geometry about the copper ion in addition to two and purple CuA. Although as much as 90% sequence difference has copper–histidine coordinations (1, 8, 9, 11, 26) (Fig. 1A). There been observed among some members of this superfamily of may also be one or two weak axial ligands depending on the proteins that span several kingdoms, sequence alignment and protein. In purple CuA proteins, such as CcO and N2OR, the phylogenic trees strongly suggest an evolutionary link and com- copper cluster is binuclear, and the copper–thiolate ligations mon ancestry. However, experimental evidence for such a link has form a diamond core Cu2S2 structure with both thiolates bridg- been lacking. We report herein the observation of pH-dependent ing the two copper ions and each copper ion additionally transformation between blue T1 copper, red T2 copper, and the coordinated by a histidine (10, 27, 28) (Fig. 1B). In the red native purple CuA centers of nitrous oxide reductase (N2OR) from copper protein NC, on the other hand, the mononuclear copper Paracoccus denitrificans. The blue and red copper centers form active site has a single copper–thiolate ligation in a distorted initially before they are transformed into purple CuA center. This tetragonal geometry that also includes two copper–histidine transformation process is pH-dependent, with lower pH resulting coordinations (13–15) (Fig. 1C). in fewer trapped T1 and T2 coppers and faster transition to purple Despite the structural differences, diverse functions, and as little CuA. These observations suggest that the purple CuA site contains as 10% sequence homology among cupredoxin proteins (25, 29, 30), the essential elements of T1 and T2 copper centers and that the CuA they share a common cupredoxin fold that adopts an eight-stranded center is preferentially formed at low pH. Therefore, this work Greek-key ␤-barrel (Fig. 1). A key difference among various provides an underlying link between the various cupredoxin cop- cupredoxin folds lies in a loop between the seventh and eighth per sites and possible experimental evidence in vitro for the strands: whereas the consensus-loop sequence for blue copper evolutionary relationship between the cupredoxin proteins. The proteins is H(x)nC(x)mH (Fig. 1A), the consensus-loop sequences findings also lend physiological relevance to cupredoxin site bio- for purple CuA and red copper are H(x)nCxxxCxxxHxxM (Fig. 1B) synthesis. and E(x)nCxxHxxxxH (13, 14) (Fig. 1C), respectively. This broad superfamily of proteins encompasses single-domain monomeric ͉ ͉ ͉ ͉ blue type 1 copper purple copper CuA red type 2 copper folding proteins, single-domain multimeric proteins, and multidomain mul- kinetics timeric proteins, suggesting that the gene encoding the fold may have duplicated or inserted itself to create the diverse structures and

upredoxins are a family of copper proteins with redox functions of cupredoxins. Sequence alignment and phylogenic CHEMISTRY Cactivities (1–3). In addition to being an important class of analyses have suggested an evolutionary link and common ancestry electron-transfer (ET) proteins (4–12), cupredoxins can be for these highly divergent proteins (25) and copper sites. However, enzymes that catalyze biological reactions (13–15). The ET experimental evidence for an evolutionary link is lacking. We cupredoxins include blue (or type 1) copper and purple CuA present herein such evidence by reporting transitions between red, proteins, whereas enzymatic cupredoxins include the red (or blue, and purple copper centers observed within the native ligand type 2) copper protein. Cupredoxins are at the heart of several donor set of a single protein, N2OR from Paracoccus denitrificans. crucial cellular processes. Aerobic and anaerobic respiration, in We also found that the transitions are pH-dependent. Mechanistic which cytochrome c oxidase (CcO) (16, 17), nitrous oxide implications are discussed as well as the significance and physio- reductase (N2OR) (18, 19), and nitrite reductase (NIR) are logical relevance to copper-site biosynthesis. actively involved, provide the cell with the energy to perform all of the essential processes that sustain life (20, 21). Iron ho- Results meostasis also has a central role in the cell to ensure that it has Red, Blue, and Purple Copper Formation in N2OR. N2OR from P. enough iron to survive, but that excess harmful iron is seques- denitrificans was cloned, expressed, and purified as metal-free tered. Ceruloplasmin, a ferroxidase, participates significantly in apo protein by using procedures described in Materials and 2ϩ these processes (22). Several cupredoxin proteins also contribute Methods. After addition of Cu ions to apo N2OR at pH 7.5 in to cellular biochemistry, such as ascorbate oxidase, nitrosocya- nin (NC), and laccase (23, 24). The cupredoxin superfamily also spans several kingdoms and, therefore, is universally important Author contributions: M.G.S., Y.E., D.K.G., and Y.L. designed research; M.G.S. and T.D.W. to organisms (25). performed research; M.G.S. and M.J.N. analyzed data; and M.G.S. and Y.L. wrote the paper. Cupredoxin names are often associated with colors, because The authors declare no conflict of interest. the proteins are intensely colored as a result of characteristics of This article is a PNAS Direct Submission. the structural features of their copper active sites. All cupre- §To whom correspondence should be addressed. E-mail: [email protected]. doxins contain copper–thiolate ligation and the thiolate-to- This article contains supporting information online at www.pnas.org/cgi/content/full/ copper charge transfer is responsible for the intense color of the 0711316105/DCSupplemental. protein. The difference in color arises from subtle differences in © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711316105 PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 7919–7924 Downloaded by guest on September 23, 2021 ABC

H(x)nC(x)mHH(x)nCxxxCxxxHxxM E(x)nCxxHxxxxH

Fig. 1. Cupredoxin folds with blue, purple, and red copper. (A) Blue copper azurin (Pseudomonas aeruginosa) cupredoxin fold (PDB ID code 4AZU) and consensus sequence. (Inset) Blue T1 active-site structure. (B) Cupredoxin domain of N2OR (P. denitrificans) (PDB ID code 1FWX) and consensus sequence. (Inset) Purple CuA active-site structure. (C )NC(Nitrosomonas europaea) cupredoxin fold monomer (PDB ID code 1IBY) and consensus sequence. (Inset) Red T2-type active-site structure.

50 mM Tris⅐HCl buffer, strong absorbances at Ϸ385 and Ϸ640 copper–thiolate protein in a type 2 copper superoxide dismutase nm were observed in the protein’s UV-visible (UV-vis) spec- (32–36). A transition near 640 nm is characteristic of thiolate- trum (Fig. 2) instead of transitions at 480, 540, and Ϸ800 nm for to-copper charge transfer in a distorted tetrahedral (T1) geom- a typical purple CuA site for N2OR from P. denitrificans as etry, as in that observed in blue copper proteins (4, 7, 11, 12). reported in the literature (19). The strong intensity of the After incubation, the blue and red copper transitions decreased absorbances is diagnostic of thiolate–copper charge-transfer in intensity, with the concomitant increase in the purple CuA transitions and, therefore, arises from copper binding to the transitions at 480, 540, and Ϸ800 nm; after overnight incubation, active-site cysteine residues of the CuA site (31). A transition only purple CuA-associated peaks remained. near 385 nm is typically associated with thiolate-to-copper After the initial observation, a systematic, aliquoted addition of charge transfer in a tetragonal (T2) geometry, similar to that copper was made to N2OR in universal buffer (UB), a multicom- observed in red copper from NC (15) and an engineered ponent UB system capable of buffering in a wide pH range from

Wavelength (nm) Wavelength (nm)

350 450 550 650 750 850 350 450 550 650 750 850 0.6 N2OR UB 7.9 A B 1.75 0.5

A ecnabrosbA A 0.4 ↑↓ 1.25 0.3 ↑ ↑ ↑↓ 0.75 0.2 ↑ 0.1 N2OR pH 8.1, 1 eq Cu, 100 min 0.25 N2OR pH 8.1, 1 eq Cu, overnight 0.0 C D

N2OR pH 8.1, 1 eq Cu, 100 min N2OR pH 8.1, 1 eq Cu, overnight simulation simulation

2.6 2.4 2.2 2.0 1.8 2.6 2.4 2.2 2.0 1.8 g value g value

Fig. 2. Spectroscopic data on N2OR reconstitution at pH 8.0. (A)N2OR copper titration in UB pH 7.9 (Ϯ0.1). (B) UV-vis scan of N2OR in UB pH 8.1 (Ϯ0.1) with 1 eq of added copper at 100 min and after overnight incubation. (C) EPR spectra of N2OR, UB pH 8.1, 1 eq of copper at 100 min and simulation. (D) EPR spectra of N2OR, UB pH 8.1, 1 eq of copper added after overnight incubation and simulation. EPR parameters are: 9.051 GHz, 30.0 Ϯ 0.5 K, modulation 5 gauss, 2-mW power, and 30 scans.

7920 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711316105 Savelieff et al. Downloaded by guest on September 23, 2021 A B C 0.6 ↑↓ 0.6 ↑↓ N2OR UB pH 6.0 N2OR UB pH 7.2 N2OR UB 8.8 0.5 0.5 ↑ ↑ A ecnabrosbA A A ecn A ↑↓ 0.4 ↑ ↑ 0.4

abr 0.3 ↑↓ ↑ ↑ 0.3 osbA ↑ ↑↓ ↑ ↑↓ 0.2 0.2 ↑ 0.1 0.1

0.0 0.0 350 450 550 650 750 850 350 450 550 650 750 850 350 450 550 650 750 850 Wavelength (nm) Wavelength (nm) Wavelength (nm)

Fig. 3. UV-vis data for reconstitution of 0.3 mM N2OR at various pH levels. (A)N2OR copper titration in UB pH 6.0 (Ϯ0.1). (B)N2OR copper titration in UB pH 7.2 (Ϯ0.1). (C)N2OR copper titration in UB pH 8.8 (Ϯ0.1).

Ϸ3.6 to Ϸ11.1. Fig. 2A shows the results of the titration for a 0.3 mM Copper Addition to N2OR at Various pH Levels. Subsequent to the sample of N2OR at pH 7.9 (Ϯ0.1) with copper additions made every initial observation of T1 and T2 copper transitions to CuA center, 5 min in Ϸ0.07 eq increments for a total of 2 eq. At low copper we embarked on a systematic investigation of these processes Ϸ Ϸ equivalents and short time scale, T1 (blue) and T2 (red) copper under different pH levels from 6.0 to 9.0. The apo N2OR transitions dominate and grow with added copper. As the titration protein was exchanged into UB buffer at various pH values. proceeds, the transitions associated with purple copper grow, and Slow, aliquoted additions of copper to 0.3 mM N2OR at various Ϸ the T1 and T2 copper transitions diminish. The presence of pH levels, up to 2 eq in 0.07 eq increments, were performed Ϯ isosbestic points indicates a conversion of the T1 and T2 coppers to as shown in Fig. 3. Fig. 3A shows the titration at pH 6.0 ( 0.1). The UV-vis scans demonstrate that some T1 or T2 copper the purple CuA center. transitions are observed, but they convert very rapidly to purple EPR Spectra of Trapped T1 and T2 Copper Sites. Intrigued by the copper center. Mild precipitation also occurs, which worsens unexpected formation of various copper sites within the same collected spectra over time and results in an elevated baseline, making a more thorough investigation difficult. Fig. 3B shows the native ligand set, we undertook an EPR investigation to verify titration at pH 7.2 (Ϯ0.2). Under these conditions, the protein formation of the different blue T1 and red T2 copper sites is more soluble, and a clear formation of T1 (Ϸ640 nm) and T2 suggested by the UV-vis data. The T1 and T2 sites were (Ϸ385 nm) copper transitions are observed at a low number of sufficiently long-lived at pH 8.0 to be trapped by flash freezing. added copper equivalents. As the number of copper equivalents Therefore, a single, 1-eq copper addition was made to N2OR in increases, the transitions associated with purple copper emerge Ϯ UB buffer at pH 8.1 ( 0.1), and the buffer was exchanged to with a concomitant decrease of T1 and T2 copper-associated remove unbound copper. A UV-vis scan was recorded on the peaks. Isosbestic points are present, which indicates a conversion sample before it was flash-frozen in a quartz EPR tube. The of T1 and T2 coppers to purple CuA. remainder of the N2OR sample was incubated overnight, and a Fig. 2A shows the scans collected at pH 7.9 (Ϯ0.1) after UV-vis scan was recorded the following day before a second copper addition. A greater amount of T2 copper relative to T1 EPR sample was prepared. Fig. 2B shows the UV-vis scan of copper forms initially on a very short time scale compared with N2OR with 1 eq of copper. At 100 min there is a dominance of that at pH 7.2. However, within 1 h the ratio of T2 to T1 copper T1 and T2 copper absorbance, which is mostly converted to is more constant until they begin to convert to the purple form.

purple CuA after overnight incubation at 10°C with a small The behavior of the system is very complex and suggests two pKa CHEMISTRY amount of remaining T1 and T2 coppers. values or ionizable residues: one responsible for the initial ratio The EPR spectrum of the sample at 100 min (Fig. 2C) shows of T2-to-T1 copper transition and one attributable to the ratio a signal arising from two species, one having a large hyperfine of both T1 and T2 copper to purple CuA transition. At the splitting parameter and one having a smaller hyperfine splitting increased pH of 7.9, the conversion of T1 and T2 copper to parameter. Simulation of the EPR data (Fig. 2C) provides g purple CuA is also observed as indicated by isosbestic points, but values of gx ϭ 2.0538, gy ϭ 2.0338, and gz ϭ 2.2499 for the species the final extent of reconstitution after overnight incubation is Ϫ4 Ϫ1 diminished slightly. with small hyperfine Az ϭϪ196 MHz (66 ϫ 10 cm ) and Finally, Fig. 3C shows copper titration at pH 8.8 (Ϯ0.1), which gx ϭ 1.9963, gy ϭ 2.0328, and gz ϭ 2.3051 for the species with large Ϫ4 Ϫ1 is much slower to convert to purple CuA. The initial ratio of T2 hyperfine Az ϭϪ454 MHz (152 ϫ 10 cm )[supporting information (SI) Table S1]. These g values are very similar to and T1 copper is enhanced compared with that at pH 7.9, and their amounts relative to purple Cu are greater still. Again, the literature g values for T1 and T2 copper sites as well as their A transient T1 and T2 coppers convert to Cu and, consistent with hyperfine splitting parameters (Table S2). The simulation shows A the trend established, a smaller amount of net reconstituted an almost equal contribution of T1/T2 copper sites of 0.88:1.00 purple sites result compared with that at pH levels of 6.0 and 7.2. at pH 8.1. After overnight incubation (16 h), the sample EPR reveals the Copper Equivalent and Spin Quantifications. To investigate the presence of at least two species; the large hyperfine pattern from above-mentioned transitions more quantitatively, various cop- the 100-min sample is still clearly present, but a new signal per equivalents ranging from 0.0 to 3.6 eq were added to a series appears (Fig. 2D). Simulation of the data indicates that a small of 0.3 mM apo N2OR solutions in UB buffer at pH 8.3 (Ϯ0.1). amount of both 100-min species remain combined with a new In one set of experiments, only Cu(II) as CuSO4 was added, seven-line pattern with parameters consistent with a purple CuA whereas in another series, an equimolar mixture of Cu(II) (as ϭ ϭ ϭ ϭ site: gx 2.0052, gy 2.0317, and gz 2.1806, with Az Cu(1) CuSO4) and Cu(I) as [Cu(CH3CN)4PF6] was added. On short 118.65 MHz (40 ϫ 10Ϫ4 cmϪ1) and Cu(2) ϭ 98.50 MHz (33 ϫ time scales, 5 min within copper addition (Fig. S1 G and H, filled 10Ϫ4 cmϪ1)(Table S1). The relative amounts of T1, T2, and circles), the amount of purple increases with increased amounts purple coppers are Ϸ1:1:1. of added copper at all equivalents investigated. However, at 5

Savelieff et al. PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 7921 Downloaded by guest on September 23, 2021 min, there are still significant amounts of blue and red sites. As the site matures, it needs to accept an electron for both When the samples are allowed to incubate (Fig. S1 G and H, Cu(II) atoms to form the Cu(1.5)Cu(1.5) mixed-valance delo- filled triangles and open circles), then the absorbance at 485 nm calized CuA site. Because no reductants were added, we propose plateaus or even diminishes slightly past 2 eq of added copper. that the reducing equivalents are provided by cysteine residues The results also demonstrate that the extent of reconstitution is in the protein. This may be an additional reason for the pH almost double in the presence of Cu(II)/Cu(I) compared with dependency on the rate and extent of reconstitution as a that in the presence of Cu(I) only (Fig. S1I) after overnight reflection of the pKa values of the cysteine residues or other incubation (18 h) and longer after incubation at 10°C. binding residues. To support the proposal that cysteines provide The amount of copper was also measured by using EPR spin reducing equivalents, titrations were performed with Cu(II) only quantification. Concentrated 0.9 mM solutions of apo N2OR in and Cu(II)/Cu(I). The results demonstrate that the extent of UB buffer pH 8.3 (Ϯ0.1) were treated with 2 eq of Cu(II), and reconstitution is greater when reduced copper is added. Under aliquots were flash-frozen at 1 min, at 13 min, and after these circumstances, the protein does not need to act as a incubation at 10°C for 24 h. The net concentration of added sacrificial reductant; otherwise, the likeliest cysteine residues to Cu(II) to the EPR samples was 1.0 mM. Spin integration against be sacrificed are the CuA active-site cysteines because they are a 1.0 mM standard solution of CuSO4 in 20% vol/vol glycerol closest to one another, which leaves fewer protein molecules demonstrated that the amount of EPR-detectable Cu(II) de- capable of binding copper. When Cu(II)/Cu(I) is added, twice creased over time. At 1 min there was 0.95 mM detectable the amount of protein reconstitutes after incubation compared copper, at 13 min there was 0.90 mM, and after overnight with Cu(II) addition (Fig. S1I, 18- and 71-h traces). Additional incubation there was only 0.60 mM EPR-detectable copper in evidence is provided by the EPR data in which the amount of the N2OR samples (Table S3 and Fig. S2). UV-vis scans dem- detectable copper decreases after copper incubation with N2OR. onstrated only red and blue copper formation within N2OR at Although 2 eq are added, a significant amount of free copper t ϭ 1 min and some purple formation at 13 min (hence the remains, because roughly half the N2OR molecules cannot bind lowered amount of detectable copper) consistent with copper due to cysteine being sacrificed to reduce Cu(II) to Cu(I). the amount of detected Cu(II). After overnight incubation, the These results contrast with studies reported on CcOCuA trun- amount of EPR-detectable copper is significantly lowered, and cates, which report near-complete incorporation of copper. In a UV-vis scan shows only purple CuA. those studies, copper was added to cell lysate so that other Discussion proteins could act as sacrificial reductants and subsequently purified on ion-exchange resins, which may have separated holo Cupredoxins constitute a large family of proteins whose func- from apo trunctates (41, 42). tions are very important in biological systems. They play impor- To offer further support for the role of cysteine in providing tant roles in numerous proteins central to several critical cellular the electron through cross-linking, we also ran native gels on apo processes ranging from aerobic and anaerobic respiration to protein, 5-min copper-treated, and overnight copper-treated oxidation and iron homeostasis. Sequence alignment has indi- N OR (Fig. S4). We found that the apo protein and 5-min cated evolutionary relationships among the proteins in the 2 copper-treated samples ran as one band on the gel, but the family, but experimental evidence for such a link has been overnight copper-treated sample exhibited an additional weak lacking. Previous studies using loop-directed mutagenesis have band of much greater molecular weight. Although most reducing succeeded in swapping the ligand loop from one type of cupre- equivalents would be provided by the intramolecular formation doxin to another type [e.g., from blue copper to purple CuA (37–39)] or different proteins among the same type [e.g., be- of disulfide bond within the CuA active site, a fraction could form tween plastocyanin and amicyanin loops transplanted into azurin intermolecularly. It is to this reducing mechanism that we in blue copper proteins (40)]. However, to our knowledge, no attribute the weak, higher molecular weight band. study has linked the different types of cupredoxin sites within the The presence of different copper sites within the same ligand same ligand loop. Herein, the cumulative data collected from set is intriguing. This is in contrast to site-directed mutants of CuA active sites, which result in blue, red, or otherwise altered both UV-vis scans and EPR on N2OR from P. denitrificans strongly suggest the formation of kinetically trapped T1 and T2 proteins as expected when the active-site electronic structure is perturbed (43–48). CcO truncates exhibiting transient color copper sites that slowly convert to the native purple CuA site. changes before forming the thermodynamically stable CuA site Mechanistic Implications. At pH 8.1, the T1 and T2 copper sites, have been reported but, to our knowledge, have not been within 1 h, formed almost equally, and the amounts remain investigated further (41). Native holo N2OR has also been widely approximately equal after incubation. This observation suggests investigated, and its various copper chromophores (both CuA a potential mechanism in which a site that can potentially and CuZ) and their spectroscopic properties have been reported accommodate two purple CuA ions in its native state can still as well as CuA reconstitution by using Cu(II)(en)2SO4 (49–51). accept two copper ions initially, one in a T1-like geometry and pH titrations of preformed CuA sites, as well as T1 sites, also one in a T2-like geometry. It is plausible for the site to adopt two results in a perturbation of the copper active site (41, 52–56). different copper geometries because the two positions in the Originally, copper sites were classified as T1, T2, or T3, but the active site are not exactly symmetrical with one site having an classification scheme was modified with the discovery of CuA. axial methionine ligand (Met-641) and the other one site having However, formation of T1 and T2 sites within the native ligand an axial carbonyl ligand from Trp-632 (19). Because no ferro- set of a purple CuA site indicates an intimate link between the magnetic or antiferromagnetic coupling was observed by EPR three, which transcends a rigorous classification scheme. The based on spin quantification, the initial T1 and T2 sites must be findings also suggest that the protein does play a role in more than 4 to 5 Å apart without a bridging ligand (Fig. S3). determining the final thermodynamic formation of copper sites Alternatively, each CuA ligand set may bind either a T1 or T2 site but that there is some plasticity in the initial formation of the such that they are far apart and uncoupled. On very short time sites. This may be the result of flexibility or conformational scales, relative amounts of T1 and T2 coppers vary strongly with freedom in the protein residues as they bind copper and of the pH, which may be attributable to ionizable groups that cannot be incoming copper atoms themselves, which need to desolvate determined at present or hydroxide ion from the buffer. Over from their hydration sphere as they encounter the protein active 1 h, their relative amounts stabilize and then slowly begin to site. Much work has been devoted toward understanding how convert to purple CuA. metals assist in protein folding in blue copper proteins (57), and

7922 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711316105 Savelieff et al. Downloaded by guest on September 23, 2021 purple CuA demonstrates that there is similar complexity in- have far-reaching implications for a broad spectrum of research volved at least in the ligand-binding loop. areas, from evolutionary studies to cellular biochemistry (molecular biology) and bioinorganic chemistry. Evolutionary Implications. Phylogenetic analysis and similarity in consensus sequence has already established an evolutionary link Materials and Methods between blue T1 copper, red T2 copper, and purple CuA proteins Materials. P. denitrificans (NCBI 8944) was purchased from the American Type (25, 29, 30). The observation of blue T1 copper and red T2 Culture Collection. Nutrient broth, nutrient agar plates, bactotryptone, and copper formation before their conversion to purple Cu in yeast extract were purchased from BD Biosciences. XL 1-Blue and BL-21 (DE3) A Star were purchased from Invitrogen. NdeI, EcoRI, and XhoI restriction en- native N2OR enzyme is an experimental confirmation of the zymes were purchased from New England Biolabs, and T4 DNA ligase was link. It has also been suggested that CuA is the combination of obtained from Invitrogen. Herculase II Fusion DNA polymerase and DpnI were two mononuclear blue sites, and the results herein demonstrate purchased from Stratagene; Taq polymerase was purchased from Invitrogen. that CuA does indeed contain the electronic elements of blue Oligos were purchased from Integrated DNA Technologies and purified by copper proteins, because T1 sites do form transiently within the denatured PAGE gel. Isopropyl ␤-D-thiogalactoside (IPTG) was purchased from CuA ligand loop (27, 58). Research Products International. All other reagents were purchased from Fisher, Sigma–Aldrich, or Fluka. Physiological Implications. Another important finding from this study is that T1, T2, and purple copper transitions are pH- Molecular Biology. P. denitrificans genomic DNA was isolated from cells grown in nutrient broth by using a Qiagen DNeasy tissue kit. The P. denitrificans gene dependent, with high pH resulting in trapped T1 and T2 coppers was amplified downstream of its leader sequence by PCR using forward primer followed by a very slow transition to purple CuA, especially on 5Ј-GGAATTCCATATGATGGCCAGCGGCGACGGCTCGGTC and reverse primer 5Ј- the time scale of protein synthesis. The results indicate that CuA TAGGCAGAATTCTCAGGCCTCCTTCGGCTCGAC-CAGC. Herculase II Fusion DNA is preferentially formed at low pH. CuA has only been reported polymerase was used to work with higher melting and extension temperatures to to date in a number of terminal electron acceptors in respiratory overcome the high GC content of the P. denitrificans genome. The gene was chains: CcO (16, 17), N2OR (18, 19), and the terminal oxidase cloned into pET17b between NdeI and EcoRI sites to yield the plasmid N2OR- from Sulfolobus acidocaldarius (SoxH) (59). The environment pET17b, which was transformed into XL 1-Blue. Clones were selected for gene for Cu in CcO and SoxH is acidic: CcO resides within the inner sequencing, and successfully sequenced (Figs. S5 and S6 plasmids were trans- A formed into BL-21 (DE3) for protein expression. The sequences had an additional membrane of mitochondria, whereas SoxH resides in the methionine at their N termini from a methionine encoded by the NdeI restriction periplasm of acidophiles (59). The acidity of their surrounding site. could promote the correct formation of CuA to the exclusion of

trapped red and blue sites and thus guarantee correct function- Protein Purification. N2OR was grown in BL-21 (DE3) cells grown in LB media ing of the enzyme. However, this would not explain the CuA and induced with 1 mM IPTG. The cells were French-pressed, and the super- formation in N2OR, which is a periplasmic protein from a broad natant was applied to a crude DEAE CL-6B column equilibrated with 50 mM ⅐ spectrum of bacteria and most likely resides at close to neutral Tris HCl/100 mM NaCl (pH 8.0) and eluted with 1 M NaCl buffer. N2OR-rich pH (20). To counter problems such as misincorporation of metal fractions were applied to phenyl Sepharose 6 FF (low sub), equilibrated with 25% (NH4)2SO4/100 mM Tris⅐HCl (pH 8.0) and eluted over 10 column volumes sites, cells have evolved chaperone proteins. Chaperones for CuA (CV) with 100 mM Tris⅐HCl (pH 8.0). N2OR-rich fractions were then applied to assembly in CcO (60) and N2OR (61, 62) have been reported. a size-exclusion column for the final purification step on small-scale purifica- Although most chaperone-bound copper is in the Cu(I) oxida- tions or to another DEAE column on large-scale purifications [conditions: 30 tion state, some chaperones believed to be involved in CuA CV from 50 mM Mops (pH 7.0) to 50 mM Mops/650 mM NaCl (pH 7.0)]. N2OR assembly have been implicated in binding Cu(II) as a physio- fractions were dialyzed against 25 mM Tris⅐HCl/100 mM NaCl and exchanged logically relevant state (63). Therefore, the observations re- into various pH UB buffer, which is a mixed five-component buffer system of ported herein for copper in the ϩ2 oxidation state may have 50 mM NaOAc and 40 mM each in Mes, Mops, Tris, and CAPS. Fig. S7 shows some physiological relevance. The findings not only underline electrospray ionization MS of the protein and a denatured PAGE gel. the importance of chaperones in shuttling copper to a site in an environment devoid of free copper but also demonstrate the Titration Experiments. N2OR samples were thawed, and copper titrations were CHEMISTRY performed on Ϸ0.3 mM protein samples with 1-␮l additions of 2.0 mM CuSO4. importance of chaperones in preventing the formation of UV-vis titrations were performed on a Cary 5000 with a mounted Peltier trapped intermediates at pH levels that may otherwise result in temperature-control unit in quartz microcells. Scan conditions were: starting inhibited enzyme function. The results also demonstrate the wavelength 900 nm, ending wavelength 200 nm, scan rate 600 nm/min with importance of chaperone proteins in providing reducing equiv- 1-nm data interval, carried out in double-beam mode, and blanked against alents from small-molecule cofactors as opposed to allowing the pH-adjusted buffer. Experiments were performed at 10°C. Argon gas was proteins they metallate to become reduced sacrificially. blown over the exterior cuvette surface to prevent water condensation, which could lead to scatter in collected spectra. Conclusions All three types of cupredoxin sites, blue type 1 copper, red type 2 EPR Experiments. EPR samples were prepared within quartz tubes to 25% final glycerol concentration as glassing agent. EPR spectra were collected at Ϸ30 K cooper, and purple CuA center, have been observed in the single- at X-band on a Varian E-122. Parameters were: field center 3,100, field sweep protein N2OR containing the native ligand donor set of a purple 1,600, power 2 mW, modulation 5 G, 30 scans, and 60 s/scan with 6 s between CuA center. After copper addition, the blue and red copper centers scans. form initially before they are transformed into purple CuA center. The higher the pH, the more blue and red copper centers form and ACKNOWLEDGMENTS. We thank Prof. Raven Huang for use of his laboratory’s the slower the conversion to purple CuA. These observations facilities and Nenad Cicmil, Weixue Wang, and Shirley Tan for general help. provide experimental evidence for evolutionary links among dif- We also thank Furong Sun (University of Illinois at Urbana–Champaign Mass Spectrometry Laboratory) for help in collecting electrospray-ionization MS ferent cupredoxins and point to the importance of pH in regulating spectra. This material is based on work supported by National Science Foun- the formation of each type of cupredoxin. The phenomenon could dation Award CHE 05-52008.

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