Abstract
The P1B-type ATPases couple the energy of ATP hydrolysis to metal ion translocation across cell membranes. Important for prokaryotic metal resistance and essential metal distribution in eukaryotes, P1B-ATPases are divided into subclasses on the basis of their metal substrate specificities. Sequence analysis of putative P1B-5-ATPases, for which the substrate has not been identified, led to the discovery of a C-terminal soluble domain homologous to hemerythrin (Hr) proteins and domains. The Hr domain from the Acidothermus cellulolyticus P1B-5-ATPase was cloned, expressed, and purified (P1B-5-Hr). P1B-5-Hr binds two iron ions per monomer and adopts a predominantly helical fold. Optical absorption features of the iron-loaded and azide-treated protein are consistent with features observed for other Hr proteins. Autooxidation to the met form is very rapid, as reported for other prokaryotic Hr domains. The presence of a diiron center was confirmed by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) data. The occurrence of a Hr-like domain in a P-type ATPase is unprecedented and suggests new regulatory mechanisms as well as an expanded function for Hr proteins in biology.
The P-type ATPases are a large family of integral membrane proteins that couple the energy of ATP hydrolysis to the transport of cations across cell membranes (1). Named for the formation of a phosphorylated intermediate during catalysis, P-type ATPases are classified on the basis of their substrate specificities and include the Ca2+, Na+/K+, and H+-ATPases (2). Members of the P1B subgroup transport transition metal ions, including Zn2+/Cd2+/Pb2+ (3-5), Cu2+ (6), Cu+/Ag+ (7), and Co2+ (8). Widely distributed in nature, the P1B-ATPases confer heavy metal tolerance to microorganisms (9) and are essential for the absorption, distribution, and bioaccumulation of metal micronutrients by cyanobacteria (10) and eukaryotes (11, 12). In humans, mutations in the Cu+ ATPases ATP7A and ATP7B lead to Menkes syndrome and Wilson disease, respectively (13).
All P1B-ATPases have the same core architecture consisting of at least six transmembrane (TM)1 helices, a soluble ATP binding domain (ATPBD), and a soluble actuator domain (A-domain), both located in the cytoplasm (Figure 1). The ATPBD includes the nucleotide binding site and a conserved DKTGT motif, of which the aspartic acid residue is phosphorylated in the catalytic cycle. Residues in the last three TM helices are proposed to form the metal binding site(s), and specificity is proposed to derive from the identities and positions of coordinating residues within these helices (14, 15). In particular, a three-residue cysteine-containing sequence motif in TM helix 4 or 6 is important for metal transport activity (16, 17). On the basis of sequence analysis and experimental evidence, the P1B-ATPases have been further divided into five substrate-specific subfamilies, designated P1B-1 through P1B-5 (14, 15).
Most P1B-ATPases include two additional TM helices as well as one or more soluble cytoplasmic metal binding domains (MBDs) at the N- and/or C-termini. Many MBDs from P1B-1- and P1B-2-ATPases exhibit a conserved ferredoxin-like fold and bind metal ions via conserved CXXC sequence motifs (18). Other types of histidine- and cysteine-rich MBDs found in the P1B-3- and some P1B-2-ATPases have also been shown to bind metal ions (19). A large body of data indicates that the MBDs play a regulatory role in ATPase function (19, 20). The P1B-4- and P1B-5-ATPases are the least well characterized and are distinguished by their relatively simple architecture, including only six TM helices (Figure 1) and an apparent absence of soluble MBDs (15). Whereas the P1B-4-ATPases are associated with transport of Co2+ and other divalent metal ions (8, 21), the substrate for the P1B-5 -ATPases has not yet been identified.
Here we report the identification, isolation, and characterization of a soluble domain found at the C-terminus of P1B-5-ATPases. Surprisingly, this domain is homologous to hemerythrins (Hr), a family of proteins and domains characterized by the presence of a carboxylate-bridged diiron center housed within a four-helix bundle (22). Characterization of this Hr-like domain from the P1B-5-ATPase of Acidothermus cellulolyticus, a gram-positive cellulolytic thermophile, reveals the presence of a diiron center. The presence of a Hr-like domain in a P1B-ATPase is unprecedented and provides insight into P1B-5 -ATPase function and the role of Hr proteins in biology.
MATERIALS AND METHODS
Bioinformatic Analysis and Sequence Alignments
Five P1B5-ATPase sequences identified previously (14) were used as an initial query using protein-protein PSI-BLAST (23) (blastp) searches against the NCBI database. Both InterPro (24) and Pfam (25) analyses of many of the sequences indicated the presence of a C-terminal Hr/HHE (histidine-histidine-glutamate) cation binding domain (PF01814, IPR012312). Sections of the Streptomyces coelicolor P1B-5-ATPase sequence (UniProtKB Q9RJ01), including the fourth TM helix and the start of the ATPBD as well as the C-terminal Hr domain, were then used for further queries. Sequences were aligned by the ClustalW method (26) with manual adjustments to ensure the best possible alignment of conserved metal binding residues. Secondary structure predictions were performed using the SOPMA Secondary Structure Prediction Method (27). The TopPred server (28) was used to predict the number and location of TM helices and SignalP 3.0 (29) was used to identify signal peptide cleavage sites
Cloning and Expression of a P1B-5-ATPase Hr domain
The gene sequence encoding the C-terminal domain (residues 620–775) of the Acidothermus cellulolyticus 11B (30) P1B-5-ATPase (YP_871788) was PCR amplified from genomic DNA (ATCC) with PCR Master Mix (Fermentas) using the primers 5’-CTGGTCGGTCTCGAATGCTGCCGGGTACGCGACAC-3’ and 5’-CTGGTCGGTCTCGGCGCTGCGCTGACCGGCATCCGGTAG-3’ (IDT), which introduce BsaI restriction sites. This sequence was selected by aligning the amino acid sequence of A. cellulolyticus P1B-5-ATPase with that of DcrH-Hr (Figure 2), the C-terminal domain of a Desulfovibrio vulgaris chemotaxis protein and the best characterized prokaryotic Hr domain (31). The homologous region begins at Leu 620 and continues to Arg 775, but does not include the final 19 amino acids of the P1B-5-ATPase. Secondary structure predictions suggest that these final residues are random coil whereas the rest of the sequence is predicted to be helical, as is characteristic of Hr domains (22, 32).
The purified PCR product and the plasmid pPR-IBA1 (IBA) were digested with BsaI (New England Biolabs), purified, and ligated, creating a construct containing an eight residue streptactin tag with a two residue linker (SAWSHPQFEK) fused to the C-terminus of the protein. The vector was transformed into E. cloni 10G chemically competent cells (Lucigen) following the manufacturer's protocol and spread on a Luria Bertani (LB) plate containing 100 μg/mL ampicillin. DNA sequencing of individual colonies confirmed the presence and accuracy of the gene fragment encoding the Hr-like domain, designated P1B-5-Hr.
For protein expression, BL21(DE3) E. coli chemically competent cells were transformed with the plasmid encoding P1B-5-Hr. Baffled flasks containing 1 LB media supplemented with 100 mg ampicillin were inoculated with an overnight culture of cells and grown at 37 °C with shaking. Protein expression was induced by adding 1 mM IPTG at an OD600 of approximately 0.6. Cells continued to grow for an additional 3–4 h, after which they were harvested by centrifugation at 4800g for 10 min in a Sorvall SLC 4000 rotor at 4 °C and stored at -80 °C.
Protein Purification
All purification and handling steps were conducted at 4 °C. The cells were resuspended in a buffer containing 50 mM Tris, pH 7.0, 200 mM NaCl, 0.5 mM PMSF and lysed by sonication for 8 min by using 20 s pulses with a 40 s rest period between each pulse. Insoluble cell debris was removed by ultracentrifugation at 120,000g in a Beckman TI-60 rotor for 1 h. The supernatant was loaded onto streptactin resin (Qiagen) and washed with four column volumes of a 50 mM Tris, pH 7.0, 200 mM NaCl wash buffer. The protein was then eluted with 1.5 column volumes of wash buffer supplemented with 2.5 mM desthiobiotin. The eluted sample (as-isolated P1B5-Hr) was concentrated in an Amicon 15 mL spin concentrator with a 10 kDa filter and its concentration was measured by the Lowry Assay (33) using bovine serum albumin as a standard.
Metal Loading and Analysis
The metal content was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) using a Varian Vista MPX ICP-OES in the Integrated Molecular Structure Education and Research Center (IMSERC) at Northwestern University. As-isolated samples were digested in 5 mL of 5% Trace SELECT nitric acid (Sigma Aldrich) in chelexed water and then filtered if necessary. Standards of iron, chromium, nickel, cobalt, zinc, copper, molybdenum, manganese, vanadium, and cadmium (Sigma Aldrich) were prepared in 5% nitric acid as well. Iron loading was performed by the aerobic addition of two equivalents of Fe(NH4)2(SO4)2·6H2O in 100 mM MES acid, pH 3.0, and 200 mM ascorbic acid to the protein solution followed by gentle mixing for 30 min. Protein was then exchanged into a buffer containing 25 mM Tris, pH 7.0, 100 mM NaCl using a 10DG desalting column (Bio-Rad). The protein and metal concentrations were measured by the Lowry assay and ICP-OES, respectively. To test whether P1B5-Hr binds metals other than iron, an apo sample was prepared by adding 10 molar equivalents of desferrioxamine (Sigma Aldrich) followed by desalting. Five equivalents of different metal ions (MnCl2, Fe(NH4)2(SO4)2, CoCl2, NiCl2, CuCl2, Zn(OAc)2, or CdCl2) were added to this apo P1B5-Hr, followed by desalting and Lowry and ICP-OES analysis..
Analytical Gel Filtration Chromatography
Analytical gel filtration chromatography was performed on a 22.5 mL Superdex 75 (GE Healthcare) column equilibrated with 50 mM Tris, pH 7.0, and 200 mM NaCl. 200 μL of either as-isolated or iron-loaded P1B-5-Hr were loaded onto the column and molecular masses were determined using the following standards: blue dextran (void volume); aldolase, 158 kDa; ovalbumin, 43 kDa; chymotrypsin, 25 kDa; and RNAse A, 13.7 kDa.
Optical and Circular Dichroism Spectroscopy
All spectra were collected at room temperature. The UV-visible spectra of P1B-5-Hr (500 μM) were recorded using a Perkin Elmer LAMBDA 1050 spectrophotometer. The azide adduct was generated by adding 50 molar equivalents of NaN3 per diiron center and incubating for 20-30 min prior to spectroscopic measurements. Reduced and oxidized samples were obtained by adding 10 molar equivalents of NaS2O4, and 1 molar equivalent of K3Fe(CN)6 per diiron center, respectively, and gently mixing immediately before spectroscopic measurements. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectrometer using a 0.6 mm path length quartz cuvette and protein diluted to 100 μM concentration in 50 mM Tris, pH 7.0, 200 mM NaCl. An average of 3 scans was collected at 20 °C with 1 nm resolution.
Thermal Stability Assay
Apo and iron-loaded P1B-5-Hr were concentrated to 2-5 mg/mL and mixed with thermal stability buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) and 5000x SYPRO Orange (Molecular Probes, Inc) in DMSO in a 25:120:2 ratio. 1 μL of each solution was distributed into each well of a PCR plate. The wells were then filled with 10 μL of buffer containing 50 mM Tris, pH 7.0 and 200 mM NaCl with some wells containing 5 mg/mL desferrioxamine as well. The plates were sealed and transferred to a Biorad CFX384 Real-Time PCR detection system in the High Throughput Analysis Laboratory (HTAL) at Northwestern University. The temperature of the plates was increased at a rate of 0.5 °C per min over the range of 10 °C to 95 °C and fluorescence was monitored at 570 nm. The data were processed using OriginPro 6.1 for Windows. In order to determine the inflection point of the melting curves, which was assumed to equal the melting temperature (Tm), a Boltzmann sigmoidal equation was fitted to the raw data. This experiment was performed in replicate up to 20 times to ensure Tm precision.
Electron Paramagnetic Resonance (EPR) Spectroscopy
Samples for EPR were concentrated to 1 mM protein in 25 mM Tris, pH 7.0, 100 mM NaCl, 20% glycerol, transferred to Q-band EPR tubes, frozen, and stored in liquid nitrogen. Cryoreduction and signal quantitation were carried out as described previously (34). Annealing at 260 K was performed by placing the EPR sample in a salt water bath and then freezing in liquid nitrogen (35). X-band spectra were collected at 10 K using a Bruker ESP300 spectrometer with the field modulation set to 10 G.
X-ray Absorption Spectroscopy
For X-ray absorption spectroscopy (XAS), samples were prepared in 25 mM Tris, pH 7.0, 100 mM NaCl, 30% glycerol with iron concentrations in the 1-2 mM range. Two independent replicates of iron-loaded P1B-5-Hr and P1B-5-Hr in the presence of 50 molar equivalents of NaN3 were prepared. Samples were loaded into Lucite cells wrapped with Kapton tape and frozen in liquid nitrogen. XAS data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 7-3, equipped with a single rhodium-coated silicon mirror and a Si[220] double crystal monochromator detuned 50% for harmonic rejection. Samples were maintained at 10 K using an Oxford Instruments continuous-flow liquid helium cryostat. Protein fluorescence excitation spectra were collected using a 30-element Ge solid-state array detector. XAS spectra were measured as described previously (36). Data were processed using the Macintosh OS X version of the EXAFSPAK program suite integrated with the Feff v7.2 software for theoretical model generation. XAS data reduction and analysis were performed following previously reported protocols (37). EXAFS data were simulated over a k range of 1 to 14.2 Å-1 for a spectral resolution of 0.12 Å. Data were fit using a scale factor of 0.95 and Eo values for Fe-O/N/C and Fe-Fe interactions of -10 and -15 respectively. Simulation parameters for fitting the raw unfiltered data are given along with the number of degrees of freedom weighted simulation “goodness of fit” (F’) parameter in Table 1.
Table 1.
Sample | Fit # | Fe-Nearest Neighbor Ligandsa | Fe-Long Range Ligandsa | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Atomb | R(Å)c | C.N.d | σ 2e | Atomb | R(Å)c | C.N.d | σ 2e | F'f | ||
Fe | 1 | O/N | 1.97 | 2.5 | 4.7 | C | 3.03 | 1.0 | 2.6 | 0.30 |
O/N | 2.09 | 2.5 | 4.6 | C | 3.46 | 2.5 | 1.3 | |||
2 | O/N | 1.97 | 2.5 | 4.91 | C | 3.02 | 1.0 | 5.5 | 0.27 | |
O/N | 2.10 | 2.5 | 5.3 | Fe | 3.39 | 1.0 | 4.8 | |||
3 | O/N | 1.97 | 2.5 | 4.9 | C | 3.02 | 1.0 | 5.7 | 0.25 | |
O/N | 2.10 | 2.5 | 5.4 | Fe | 3.39 | 1.0 | 4.7 | |||
C | 4.09 | 1.0 | 4.9 | |||||||
Azide | 1 | O/N | 1.99 | 2.5 | 4.3 | C | 3.04 | 1.0 | 2.0 | 0.21 |
O/N | 2.12 | 2.5 | 4.1 | C | 3.46 | 3.5 | 2.8 | |||
2 | O/N | 1.99 | 2.5 | 5.0 | C | 3.03 | 1.0 | 3.5 | 0.16 | |
O/N | 2.11 | 2.5 | 5.8 | Fe | 3.39 | 1.0 | 4.3 | |||
3 | O/N | 1.99 | 2.5 | 5.0 | C | 3.03 | 1.0 | 3.3 | 0.14 | |
O/N | 2.11 | 2.5 | 5.7 | Fe | 3.39 | 1.0 | 4.2 | |||
C | 4.12 | 1.0 | 3.0 |
Independent metal-ligand scattering environment
Scattering atoms: O (Oxygen), N (Nitrogen), C (Carbon) and Fe (Iron)
Metal-ligand bond length (all standard deviations < 0.03 Å)
Metal-ligand coordination number (all standard deviations < 1.0)
Debye-Waller factor given in Å2 × 103 (all standard deviations < 0.9 Å)
Number of degrees of freedom weighted mean square deviation between empirical and theoretical data
RESULTS AND DISCUSSION
Sequence Characteristics of Hr Domain-Containing P1B-5-ATPases
A BLAST search using a 60-residue sequence from the S. coelicolor P1B-5-ATPase, one of the previously identified P1B-5-ATPases (14), corresponding to the fourth TM helix and the start of the ATPBD yielded 195 complete P1B-5-ATPase sequences. This residue span was selected because it includes residues in the putative TM metal binding site proposed to confer substrate specificity, including the cysteine- and proline-containing signature motif. It is therefore ideal for distinguishing members of the P1B-5-ATPase subfamily. Every species containing a P1B-5-ATPase is eubacterial, but beyond this broad classification, there are no obvious unifying characteristics. P1B-5-ATPases are found in both gram positive, gram negative, aerobic, anaerobic, facultative, and microaerobic organisms. Of the 195 identified sequences, 40 contained Hr-like domains. Membrane topology predictions indicate the presence of six to seven TM helices. Those proteins that have seven predicted TM helices, including the A. cellulolyticus P1B-5-ATPase, contain a signal peptide cleavage site between the first two helices, consistent with a six-helix architecture. The P1B-4-ATPases are also predicted to have six TM helices, and these six helices correspond to the final six helices of the eight helix P1B-1-, P1B-2, and P1B-3-ATPases (15).
With a larger library of sequences identified, the defining characteristics of the P1B-5 subfamily (14) can be refined (Figure 1). Alignment of the 195 P1B-5-ATPase sequences (Supplemental Figure 1) indicates that the signature sequence in the fourth TM helix is (T/S)PCP with threonine present in 113 sequences and serine in the other 82 sequences. In 143 of 195 sequences, the fifth TM helix begins with a glutamine. The fifth helix also contains a conserved potential metal binding residue near the periplasmic face, a methionine in 171 sequences and a glutamic acid in 24 sequences. The final TM helix houses a QEXXD motif that is conserved in all but two P1B-5-ATPases. This motif along with the (T/S)PCP sequence likely contributes key substrate binding residues. Previously noted serine and asparagine residues in the fifth and sixth helices, respectively (14), are not universally conserved in this more extensive sequence library.
Like most P1B-4-ATPases, the P1B-5-ATPases lack N-terminal soluble MBDs, suggesting that the two N-terminal TM helices in the other subfamilies may serve to tether and position the MBDs for regulatory interactions with the other soluble domains. The only exception, from Burkholderia glumae, contains a membrane-bound cytochrome b561-like domain (PF01292, IP011577) fused to the N-terminus. The P1B-5-ATPases are common to the Burkholderia genus, and a gene encoding a cytochrome b561-like protein is sometimes found in the same operon (e.g. B. cenocepacia J2315, B. vietnamiensis G4, B. ambifaria MC40-6, B. multivorans ATCC 17616). In some organisms, these cytochrome b561 proteins are associated with NiFe hydrogenases (38, 39), although this enzyme does not appear to be present in all Burkholderia genomes encoding a P1B-5-ATPase.
The only C-terminal soluble domain found in the P1B-5-ATPases is the Hr-like domain. Interestingly, all 40 Hr-containing P1B-5-ATPases have the conserved methionine in the fifth TM helix, and 38 have a TPCP signature motif in the fourth helix. These Hr domains were not identified in recent studies of Hr diversity and classification (40, 41), probably due to the low overall sequence homology. For example, A. cellulolyticus P1B-5-Hr is 18% identical and 40% similar to DcrH-Hr. Moreover, the metal binding residues are not completely conserved. In structurally characterized eukaryotic and prokaryotic Hr proteins, the two iron ions are bridged by an aspartic acid and a glutamic acid and coordinated by three and two histidines, respectively (22, 32) (shown for DcrH-Hr in Figure 2). In 27 of 40 sequences, the residue corresponding to DcrH-Hr Asp 946 (numbering based on the full length DcrH sequence) is glutamic acid and in 23 sequences, aspartic acid replaces DcrH-Hr Glu 886. Moreover, one of the metal coordinating histidine residues, equivalent to DcrH-Hr His 905, is completely absent in all the P1B-5-ATPase Hr domains (Figure 2). Some of the identified P1B-5-Hr sequences have a histidine one amino acid later, yielding HX4H rather than HX3H, but this only occurs in 7 of the 40 sequences, suggesting it may not be involved in metal binding. There is a conserved glutamic acid two amino acids prior to the histidine position. Thus, the consensus motif of P1B-5-Hr is H– HX3(D/E)–HXE–HX4(E/D). In A. cellulolyticus P1B-5-Hr, these residues are His 644, His 685, Glu 689, His 714, Glu 716, His 757, and Asp 762. Alterations in the metal binding residues are found in a large number of predicted prokaryotic Hrs (41). In addition, there are significant deviations between the hydrophobic substrate channel residues of classical Hr domains and the corresponding residues in the P1B-5-Hr proteins. Of the 21 residues that comprise the substrate channel in DcrH-Hr (32), only seven are similar in P1B5-Hr proteins.
Isolation and Characterization of P1B-5-Hr
Overexpression of P1B-5-Hr yielded approximately 3 mg purified protein per 1 L of media. The single-step purification on the streptactin column was sufficient for >95% purity (Figure 3). Samples were analyzed for a range of metal ions by ICP-OES, and only iron was detected. The iron stoichiometry was variable, with values ranging from 0 to 2 iron ions per protein monomer. If the stoichiometry was less than 2 iron ions per protein, two equivalents of Fe(NH4)2(SO4)2·6H2O were added and then excess iron was removed on a desalting column. After this treatment, the stoichiometry was 2.13 ± 0.10 iron ions per protein. An apo form of P1B-5-Hr was prepared by desferrioxamine treatment and then loaded with five equivalents of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II). Stoichiometries of 0.22 ± 0.01 Mn(II), 1.93 ± 0.06 Fe(II), 0.75 ± 0.02 Co(II), 0.48 ± 0.03 Ni(II), 1.10 ± 0.01 Cu(II), 1.25 ± 0.12 Zn(II), and 0.92 ± 0.02 Cd(II) were measured. Given the consistent measurement of 2 Fe per protein and the spectroscopic data (vide infra), it seems likely that P1B-5-Hr binds iron specifically and perhaps binds other metal ions adventitiously.
The oligomeric state of P1B-5-Hr in solution was investigated by analytical gel filtration experiments (Figure S1). The as-isolated sample eluted in two peaks, one at ~34 kDa and a second beyond the calibration range but below the void volume (~200 kDa). The theoretical mass of P1B-5-Hr, including streptactin tag and linker is 18.5 kDa, consistent with the presence of a dimer and a higher molecular mass oligomer. The same two peaks are observed for the iron-loaded sample, although with slightly altered calculated masses of ~40 kDa and > 300 kDa. In addition, an aggregate peak is present, consistent with some precipitation observed upon iron addition.
CD and Optical Spectroscopy
The CD spectra of both apo and iron-loaded P1B-5-Hr exhibit local minima at 209 nm and 221 nm (Figure 4), indicative of a primarily helical structure, consistent with a Hr four-helix bundle (22). The similarity between the CD spectra of the iron-loaded and apo samples suggests that iron binding does not induce major structural changes. The purified, iron-loaded protein is always yellow in color and its optical spectrum (Figure 5) lacks a ligand-to-metal charge transfer (LMCT) band observed at 500 nm for the oxy form of Hr, which has a terminally bound hydroperoxide ligand (31, 42, 43). Reduction by excess dithionite bleached the yellow color, which reappeared upon exposure to air, without forming any detectable absorbance features attributable to the oxy form, indicating that autooxidation to the met form is very rapid (t1/2 < 1 min). Rapid autooxidation was also reported for the prokaryotic Hr domain DcrH-Hr (31) and Hr from Methylococcus capsulatus (Bath) (42). A shoulder at approximately 650 nm is a d-d transition from the diiron(III) site (44). There is a broad absorbance in the 300-400 nm range attributable to LMCT transitions, but distinct peaks are not observed as in other Hr proteins and domains (31, 42, 44, 45). Addition of the oxidizing agent potassium ferricyanide did not create any new spectroscopic features (data not shown) indicating that the iron-loaded sample contains only Fe(III). The spectrum of the Hr domain from the human FBXL5 protein is similar (46). Interestingly, alignments indicate that the histidine residue absent in P1B5-Hr is also missing in FBXL5. Thus, the broader spectroscopic features in the 300-400 nm range may be due to differences in iron coordination in P1B-5-Hr. Addition of azide produces a new optical feature at 453 nm (Figure 5). Similar features are observed for DcrH-Hr (31), M. capsulatus (Bath) Hr (42), and Hr from marine invertebrates (44, 47).
Thermal Stability Assay
To evaluate the effect of iron loading on P1B-5-Hr stability, thermal stability assays were performed. SYPRO Orange, a dye that fluoresces when interacting with hydrophobic residues and can act as an indicator for denatured protein, was mixed with both apo and iron-loaded P1B-5-Hr. These solutions were gradually heated while the fluorescence was monitored, and the signal normalized to generate a denaturation curve (Figure 6). Fitting of these curves yields Tm values of 65.5 ± 1.5 °C for apo P1B-5-Hr and 74.9 ± 2.0 °C for iron-loaded P1B-5-Hr. To verify that the increased thermostability is a direct effect of the iron bound to P1B-5-Hr, conditions containing an excess of desferrioxamine were tested for thermal stability. Apo protein was relatively unaffected by the desferrioxamine with a Tm value of 65.8 ± 0.2 °C. The iron-loaded protein, however, exhibited a decreased Tm to 63.4 ± 0.3 °C, effectively equivalent to the apo sample. The Tm of the apo protein correlates well with temperature-dependent growth of A. cellulolyticus which has a maximum growth temperature of 65 °C.
EPR Spectroscopy
Iron-loaded P1B-5-Hr is EPR silent, consistent with an antiferromagnetically coupled diiron(III) center. Initial attempts to generate an EPR signal by semi-reduction with dithionite or reduction followed by semi-oxidation were unsuccessful, perhaps due to instability of the mixed valence state. Instead, to generate a mixed valence Fe(II)Fe(III) center, a frozen, iron-loaded sample was reduced by γ-irradiation as done previously for Hr (48) and other diiron-containing proteins, including the soluble methane monooxygenase hydroxylase (34) and the ribonucleotide reductase R2 protein (48). After annealing at 260 K to eliminate radicals in the frozen matrix whose signals partly obscured that of cryoreduced P1B-5-Hr, the spectrum from the protein shown in Figure 7 was obtained. The g-values of 1.94, 1.83, and 1.76 rule out an assignment to a mononuclear iron center, and instead definitively reveal the presence of a mixed valence Fe(II)Fe(III) diiron cluster in which the partner Fe ions are antiferromagnetically coupled to generate an S=1/2 cluster state (48, 49). Quantitation with a Cu(II)-EDTA standard indicates that the dose of 1.4 Mrad created ~0.1-0.15 mM Fe(II)Fe(III) cluster. Our experience indicates that this dose typically reduces roughly 15% of the diamagnetic parent, leading to an estimated total concentration of diiron centers of ~0.75 mM, which corresponds approximately to ~ 3/4 occupancy of the sites (sample concentration 1 mM). A signal at g = 4.3 was also present, and quantitation indicates that the concentration of adventitious Fe(III) is ~0.1 mM or about 10% of the total iron.
X-Ray Absorption Spectroscopy
To confirm the presence of a diiron center, XAS data were collected on samples of iron-loaded P1B-5-Hr and P1B-5-Hr in the presence of azide. The X-ray absorption near edge spectra (XANES) indicate the presence of both Fe(II) and Fe(III) in the protein-bound Fe XANES (Figure 8). Analysis of the first inflection energies for protein-bound Fe compared to Fe(II) and Fe(II) model controls indicates that the iron-loaded sample (inflection energy of 7124.6 eV) has a 50% mixture of Fe(II) and Fe(III) whereas the azide treated sample (inflection energy of 7123.9 eV) has a 65%/35% mixture of Fe(II)/Fe(III). Immediately following exposure of the iron-loaded sample to the X-ray beam, the resulting metal appears to be a stable equivalent mixture of Fe(II) and Fe(III). The azide treated sample was highly susceptible to X-ray influenced metal photoreduction.
The extended X-ray absorption fine structure (EXAFS) data for both protein samples are best fit with multiple shells of interacting ligands (Figure 9, Table 1). There are two O/N ligands at 1.97-1.99 Å, four O/N ligands at 2.09-2.12 Å, consistent with six-coordinate iron (Figure 2) and a highly prevalent scattering Fe-Fe signal in both samples at 3.39 Å. This Fe-Fe distance is identical to the 3.39 Å distance observed crystallographically for met DcrH-Hr and longer than the 3.25 Å distance typical of invertebrate Hr (22, 50). An increase in Fe-Fe distance is consistent with the presence of a partly photoreduced diiron center (32, 51). The two nearest neighbor O/N ligand environments likely correspond to histidine nitrogen atoms and carboxylate oxygen atoms at ~2.1 Å, as observed in other Hr structures, and perhaps exogenous oxygen-based ligands at 1.97-1.99 Å. The latter distance is too long for a bridging oxygen atom and suggests the presence of a bridging hydroxide, as observed for the deoxy form of Hr (52), or alternatively, a water molecule. The azide treated sample is very similar to the iron-loaded sample, which is not surprising since the Fe-N distance in azide adducts of Hr is approximately 2 Å (32, 50). The Fe-Fe distance is unchanged, suggesting that binding of azide does not alter the dinuclear center core structure in any appreciable manner as would be expected if azide was bridging the two metal ions. Thus, azide is likely binding in a terminal fashion, as observed for other Hr adducts. Long-range carbon scattering environments at ca. 3.0 Å and 4.1 Å are observed in both samples. Carbon scattering at these bond distances is typical in the presence of rigid imidazole scattering from histidine residues acting as metal ligands (53).
Functional Implications
The combined metal binding and spectroscopic data indicate that P1B-5-Hr houses a diiron center, similar to that in other Hr proteins and domains. Hr domains have been implicated in a variety of biological functions. In invertebrates, Hr reversibly binds O2 for transport (54). Prokaryotic Hr proteins and domains fall into two groups: single domain proteins or domains fused to larger proteins. Rigorous functional characterization has not been performed, but putative functions include O2 transport (M. capsulatus (Bath) Hr) (42, 55), O2 sensing for chemotaxis (DcrH-Hr) (31), nitric oxide sensing (56), iron storage (40, 57), and cadmium detoxification (58). In humans, iron sensing by the Hr domain of FBXL5 leads to degradation of iron regulatory proteins (46, 59).
Soluble metal-binding domains in other P1B-ATPases play a regulatory role, likely via metal-dependent protein-protein interactions with other domains. For example, Cu+ binding by the N-terminal MBDs of P1B-1-ATPases precludes protein-protein interactions with the ATPBD (60, 61). Although the substrate of the P1B-5-ATPases has not been identified, the discovery of a soluble Hr domain provides intriguing clues. One possibility is that these ATPases transport iron. By analogy to the P1B-1-ATPases, iron binding to P1B-5-Hr might affect transport by modulating interdomain interactions. An alternative or additional function of P1B-5-Hr might involve O2 sensing. For DcrH-Hr, conformational changes upon O2 binding to the diiron(II) site are proposed to initiate signal transduction that leads to anaerotaxis (32). For the P1B-5-ATPases, sensing of O2 as well as iron would have the added benefit of preventing oxidative damage: under oxidative stress conditions, the iron-loaded Hr domain would initiate iron efflux, minimizing the potentially damaging effects of iron-mediated Fenton chemistry.
Another possibility is that iron binding and/or O2 sensing regulate transport of another metal ion. Notably, the A. cellulolyticus genome encodes a nickel-containing superoxide dismutase, and O2 sensing could play a role in its regulation by maintaining appropriate Ni2+ concentrations. Another potentially O2-regulated system present in A. cellulolyticus is NiFe hydrogenase (62). This idea is consistent with the observation (vide supra) that some Burkholderia species encode P1B-5-ATPases and cytochrome b561-like proteins in the same operon, and cytochrome b561-like proteins may be associated with NiFe hydrogenase (38, 39). There could also be synergism or antagonism between iron and the transported metal substrate. Although testing of these hypotheses will require functional characterization of a full length P1B-5-ATPase, the identification of P1B-5-Hr suggests a novel regulatory mechanism for P1B-ATPases and expands the known functional repertoire of Hr domains.
Supplementary Material
ACKNOWLEDGEMENTS
We thank J. Argüello for valuable discussions. We also thank Dr. Chi-Hao Luan and Brendon Dusel of the High Throughput Analysis (HTA) Laboratory at Northwestern University for help in collecting and analyzing thermal stability data.
Footnotes
This work was supported by NIH grants GM58518 (A. C. R.), DK068139 (T. L. S.), and HL13531 (B. M. H).
Abbreviations: A-domain, actuator domain of a P1B-type ATPase; ATPBD, ATP binding domain of a P1B-type ATPase; DcrH-Hr, hemerythrin domain from Desulfovibrio vulgaris chemotaxis protein; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; Hr, hemerythrin; MBD, metal binding domain; P1B-5-Hr; hemerythrin domain from the Acidothermus cellulolyticus P1B-5-ATPase; TM, transmembrane; XANES, X-ray absorption near edge spectra.
SUPPORTING INFORMATION AVAILABLE: Gel filtration data for purified P1B-5-Hr and list of 195 identified P1B-5-ATPase sequences. This material is available free of charge via the Internet at http://pubs.acs.org.
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