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Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus
Available online at www.sciencedirect.com JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 102 (2008) 480–488 www.elsevier.com/locate/jinorgbio
Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus q Mark Pluym a, Naomi Muryoi b, David E. Heinrichs b,*, Martin J. Stillman a,* b
a Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 15 October 2007; received in revised form 14 November 2007; accepted 15 November 2007 Available online 3 December 2007
Abstract Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Iron regulated surface determinant; Near iron transporter; Gram-positive bacteria; Iron acquisition; Magnetic circular dichroism spectroscopy; Heme binding protein; Electrospray ionization mass spectrometry; Heme iron spin states
1. Introduction Worldwide, Staphylococcus aureus (S. aureus) is one of the most important causes of life-threatening bacterial infections. It causes a variety of diseases ranging from mild skin and soft tissue infections to severe life-threatening infections. The rapid spread of multi-drug resistant strains, q This work was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to both D.E.H. and M.J.S.), and the NSERC Postgraduate Scholarship program (funding for M.P.). We acknowledge technical support provided by Johnson Cheung. * Corresponding authors. Tel.: +1 519 661 3984; fax: +1 519 661 3499 (D.E. Heinrichs), tel.: +1 519 661 3821; fax: +1 519 661 3022 (M.J. Stillman). E-mail addresses: [email protected] (D.E. Heinrichs), martin.stillman@ uwo.ca (M.J. Stillman).
0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.11.011
including methicillin-resistant S. aureus (MRSA), has added urgency to the development of effective treatment and prevention in those more susceptible to infection. Iron is an essential nutrient for almost all organisms, including most bacteria, but it is frequently a growth limiting nutrient due to its insolubility in the presence of oxygen and, in vivo, its sequestration by proteins both inside and outside of cells. Successful pathogens, including S. aureus, have developed mechanisms to thrive under the extreme levels of iron restriction present in the host environment. Heme iron, present within hemoglobin in red blood cells, or in myoglobin, represents the largest iron reservoir in the human body and can be a significant source of iron for some bacterial pathogens [1]. Recent studies have shown that S. aureus scavenges heme using iron-regulated surface determinant (Isd) proteins [2,3], of which four proteins, IsdA, IsdB, IsdC and
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HarA (or IsdH), are covalently anchored to the bacterial cell wall. Each possesses at least one copy of an approximately 150 amino acid residue domain referred to as a NEAT domain [2]. The widespread occurrence of the NEAT domain has led to a number of studies aimed at determining its specific function in proteins located at the Gram-positive bacterial cell surface [4–6,3]. In combination, and in agreement with previous spectroscopic studies [4–6,3], analysis of the crystal structures of S. aureus IsdA NEAT (NEAT-A) [3] and IsdC NEAT (NEAT-C) [4], have demonstrated that these particular domains coordinate one heme molecule with axial coordination of heme iron mediated by a single tyrosine residue. Based on the presence or absence of this tyrosine as well as other ‘heme-pocket’ residues [3], it is possible to subdivide NEAT domains into those predicted to bind heme and others that would not coordinate heme, at least in similar fashion. Given this, each of the four S. aureus cell-wall-anchored Isd proteins (i.e. IsdA, IsdB, IsdC and HarA/IsdH) contain one NEAT domain that binds heme. In this paper, we report studies using a combination of absorption and MCD spectroscopy and ESI-MS to elucidate details of the heme environment in the NEAT domains of IsdA (NEAT-A) and IsdC (NEAT-C). The absorption and MCD data indicate that both NEAT-A and NEAT-C bind high-spin ferric hemes. The heme binding pocket is inaccessible to small anionic ligands but reduction of the heme in NEAT-A opens access, allowing for CO axial ligation. In contrast, reduction of the heme in NEAT-C results in loss of the heme from the binding site of the protein. A comprehensive understanding of NEATdependent heme binding will aid in our ability to understand the mechanisms of heme binding and release, and possible heme transfer reactions between Isd proteins. 2. Results The NEAT domains, which are studied here are part of the sequences in the Isd cell-wall-anchored IsdA and IsdC proteins, and have been shown to function as the heme binding domains in a wide range of other heme binding proteins [4,3,7]. While spectroscopic data have been reported by us previously for IsdA [7], IsdC [8,9], and IsdE [9], there have been no MCD spectral data reported previously for isolated NEAT domains from any protein. In this study, the recombinant NEAT domains of S. aureus IsdA (NEAT-A) and IsdC (NEAT-C) proteins were used to further investigate the heme binding properties of the complete proteins, IsdA and IsdC. These studies also answer the question about how independently the NEAT domains act in binding heme in the Isd series of proteins. Previous studies have shown that IsdA efficiently scavenges heme from within Escherichia coli [7], whereas IsdC scavenges primarily PPIX [8,9]. For both proteins, the major fraction isolated following purification is the heme- or PPIX-bound holo form. Previous attempts at removing bound heme to form the apo-protein using the traditional 2-butanone
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extraction were unsuccessful because the remaining apoprotein aggregated and precipitated. However, utilizing heme-deficient E. coli proved an effective means of obtaining apo-NEAT-A and apo-NEAT-C. These species were then reconstituted with heme, which allowed for a complete spectroscopic study of the oxidation state and spin states of the heme iron bound in the NEAT-C and NEAT-A domains, as previously reported for IsdA [7]. The spectral data reported here also show that reconstituted NEAT-A exhibits heme binding characteristics almost identical to those of the whole IsdA protein, that is the heme-boundIsdA. We, therefore, infer that the heme binding properties of the NEAT-C species also represent those of the whole IsdC protein, of which there exist no extensive spectroscopic data [8]. 2.1. IsdA NEAT heme binding The mass spectrum of apo-NEAT-A, following purification from E. coli, shows a peak at 16,377 Da (Fig. 1B) that is associated with the heme-free or apo-NEAT-A protein. The relative intensities of the heme-free (16,377 Da) and hemebound (16,993 Da) peaks in Fig. 1B show that less than 10% of the protein is bound to heme. Two distributions of charge states exist in apo-NEAT-A, centered about +15 and +8 (Fig. 1A). Following the addition of hemin and purification using size-exclusion chromatography, these charge states remain unchanged, with the two distributions centered as before at +15 and +8 (Fig. 1C). Following this heme loading reaction, over 95% of the protein is observed to be bound to heme, as shown by the intensification of the peak at 16,995 Da (Fig. 1D). Furthermore only trace amounts of free heme are present (616 m/z in Fig. 1C). The fact that the observed charge states remain unchanged following heme binding suggests that little conformation change occurs as a result of heme binding [10,11]. This is in complete agreement with crystal structure data for apo and holo NEAT-A that was recently reported [3]. As described above, a small amount of heme was bound to NEAT-A following purification of the predominantly apo-NEAT-A from E. coli, as shown in Fig. 1B. The corresponding absorption spectrum is shown in Fig. 2A, which exhibits the characteristic bands of a high-spin ferric heme, in particular the charge transfer band at 628 nm [7,12,13]. The strong intensity of the 277 nm protein band in relation to the Soret band indicates the relative abundance of apoNEAT-A as observed in the mass spectrum. As expected, following heme loading the Soret band becomes much more intense in relation to the 276 nm protein band. The absorption and MCD data presented in Fig. 3A show that like whole IsdA protein, the iron in heme-bound-NEAT-A is a 5-coordinate, high-spin, Fe(III) heme, which, based on our previous MCD studies and the results of X-ray diffraction analysis, is known to be coordinated by a tyrosine residue [7,12]. Axial ligation by cyanide in the ferric heme state is restricted (Fig. 2B) as reported for the whole IsdA protein. However, following reduction
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Fig. 2. Absorption spectra of NEAT-A (A) following purification from E. coli and (B) following the addition of hemin.
2.2. IsdC NEAT heme binding
Fig. 1. Charge state and mass spectra of apo-NEAT-A and holo-NEATA. (A) Charge state spectrum of NEAT-A following purification from E. coli. (B) Deconvoluted mass spectrum of NEAT-A showing apoNEAT-A (16,377 Da) and holo-NEAT-A (16,993 Da). (C) Charge state spectrum of NEAT-A following the addition of hemin. (D) Deconvoluted mass spectrum of NEAT-A following the addition of hemin showing apoNEAT-A (16,379 Da) and holo-NEAT-A (16,995 Da).
(Fig. 4A) access was possible for carbon monoxide, which binds strongly to the Fe(II), dramatically changing the optical spectra (Fig. 4C) [7,14]. From this we can conclude that the protein regions outside of the NEAT domain of IsdA play little role in heme binding, most likely acting as a scaffold for the NEAT domain. The data presented here indicate that NEAT-A has ligand binding properties like IsdA.
Previous reports on the heme binding properties of IsdC were complicated due to the predominance of PPIX following isolation from E. coli [8]. While a small fraction of heme was bound to the IsdC as isolated, the optical spectra were dominated by the presence of the PPIX. In this work, we now can report the heme binding properties of apoNEAT-C following reconstitution with hemin. The presence of the peak at 16,192 Da in the mass spectral data shown in Fig. 5 clearly show that following purification the majority (95%) of the NEAT-C produced recombinantly is heme-free (Fig. 5B). Significantly, there is no indication that free PPIX is in solution, because no band at 564 m/z is observed [8]. Following hemin loading, approximately 95% of the protein is heme-bound (16,809 Da in Fig. 5D). The charge state spectrum of apo-NEAT-C is dominated by the +8 and +9 charge states (Fig. 5A). This distribution is unchanged following heme loading (Fig. 5C) suggesting little conformation change in the protein takes place after heme binding, a result similar to the data for NEAT-A [10,11]. Again, the lack of change in the charge states points to the value of mass spectral data in determining conformational changes upon substrate binding. As described above from the ESI-MS data, the apoNEAT-C sample included a small fraction bound to heme
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Fig. 3. Absorption and MCD spectra of ferric heme loaded NEAT-A. (A and B) Following purification and (C and D) following the addition of sodium cyanide. In this and all figures showing absorption and MCD data, the ordinate (y-axis) is scaled as DAL-R/M; that is, the MCD signal intensity is in units of Tesla 1. The ordinate for the absorption spectrum is also absorbance so that both spectra scale linearly. The absorption and MCD spectra were recorded for the same solution at the time the MCD spectrum was measured.
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Fig. 4. Absorption and MCD spectra of ferrous heme loaded NEAT-A. (A and B) Following the addition of sodium hydrosulfite and (C and D) following the addition of sodium hydrosulfite and carbon monoxide.
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Fig. 5. Charge state and mass spectra of apo-NEAT-C and holo-NEATC. (A) Charge state spectrum of NEAT-C following purification from E. coli. (B) Deconvoluted mass spectrum of NEAT-C showing apoNEAT-C (16,192 Da) and holo-NEAT-C (16,808 Da). (C) Charge state spectrum of NEAT-C following the addition of hemin. (D) Deconvoluted mass spectrum of NEAT-C following the addition of hemin showing apoNEAT-C (16,193 Da) and holo-NEAT-C (16,809 Da).
following purification of the isolate from E. coli. This small component exhibited an absorption spectrum with a Soret band at 404 nm and visible region bands at 501, 534, and 625 nm. Following heme loading and subsequent purification, a matching, but far more intense, spectrum was obtained in which the Soret band absorbance now greatly
Fig. 6. Absorption and MCD spectra of ferric heme loaded NEAT-C. (A and B) Following chromatographic separation and (C and D) following the addition of sodium cyanide.
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Fig. 7. Absorption and MCD spectra of ferrous heme loaded NEAT-C. (A and B) Following the addition of sodium hydrosulfite and (C and D) following the addition of sodium hydrosulfite and carbon monoxide.
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exceeds that of the NEAT-C residues in the 277 nm region. This result is good evidence that the heme added to the apo-NEAT-C binds in the same manner as the heme that bound in the E. coli. The MCD spectrum of NEAT-C, following the addition of excess heme and purification, is shown in Fig. 6B. This detailed spectrum is very similar to that recorded for NEAT-A. From our analysis of the absorption and MCD spectra of NEAT-A we can, therefore, conclude that NEAT-C also binds a 5-coordinate, high-spin, ferric heme through a proximal tyrosine residue [4,8,12]. The test of access to the heme iron, using cyanide, again failed and did not result in a low-spin ferric iron center, from which we conclude that axial access to the heme iron is also blocked in NEAT-C. However, there were differences between NEAT-C and NEAT-A following reduction of the heme. The addition of sodium hydrosulfite to NEATC resulted in a very complex spectrum representative of a mixture of hemes (Fig. 7). This is quite unlike the case with NEAT-A, where reduction of the heme resulted in formation of a high-spin ferrous heme, coordinated by a histidine residue, as reported for IsdA [7,14]. Unlike NEAT-A, NEAT-C does not contain a histidine residue near the distal heme site; instead, an isoleucine is present [4,3]. The bands arising from the presence of both bound ferric heme and reduced ferrous heme produce the complex absorption and MCD spectra shown in Fig. 7A. It should be noted that an approximately 2 greater concentration of sodium hydrosulfite was required to reduce the heme in NEAT-C to obtain the spectrum in Fig. 7A than that required to reduce the heme in NEAT-A. The mixture of bands in the spectra, and the high levels of reducing agent required, suggest that in fact the ferrous heme is released by NEATC and the remaining bound heme is still oxidized. We confirmed this in experiments using CO. Following the addition of sodium hydrosulfite, and then carbon monoxide, to NEAT-C, an MCD spectrum different to that of NEAT-A was recorded. Instead of a 2:1 intensity ratio between the Soret and Q band A-terms, a ratio closer to 1:1.5 was observed. Furthermore, the band positions were blue-shifted. For NEAT-C, the A-term crossover points were observed at 410 and 565 nm, whereas for NEAT-A the A-term crossover points were observed at 421 and 569 nm. As we have noted previously, the spectral patterns for different oxidation, spin and ligand states possible for heme are surprisingly consistent across many proteins, so these changes must be the result of formation of a different species. The absorption and MCD spectra of hemin in a 1:1 mixture of DMSO and 1 PBS, and following the addition of sodium hydrosulfite and carbon monoxide, are shown in Fig. 8. The bands observed are very similar to those of NEAT-C following the same chemistry (Fig. 7B), with MCD crossover points at 410 and 563 nm. We propose then that following reduction, NEAT-C releases bound heme and upon addition of carbon monoxide, the heme becomes coordinated to CO.
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Fig. 8. Absorption (A) and MCD (B) spectra of bis-CO ferrous heme in a 1:1 mixture of DMSO and 1 PBS.
3. Discussion The bacterial iron-regulated Isd proteins were first identified in S. aureus [15] but are also present in other Gram-positive bacteria, such as Listeria monocytogenes and Bacillus anthracis. A subset of the Isd proteins, IsdA, IsdB, IsdC and IsdH, are proposed to take part in the extracytoplasmic membrane transfer of heme from host serum heme-proteins through to membrane-localized heme transporters. Our initial studies of the heme binding properties of IsdA [7], IsdC [8], and IsdE [9] were based on recombinant proteins representative of the full length proteins, as expressed in the cytoplasm of E. coli. Both IsdA and IsdC contain a single copy of the NEAT domain, which operates as the heme binding site [4,16]. Studying the isolated NEAT domains of IsdA and IsdC has several advantages over studying the entire proteins. Most significantly, the increased stability observed for the NEAT domains in buffered solution allows for greater storage periods without any apparent aggregation or degradation and allows more detailed studies of the mechanistic properties of these proteins. Focusing strictly on the NEAT domains of the proteins, however, assumes that the removed portions of the proteins (see Experimental) have no or insignificant effect on the heme binding properties of the holo-proteins. The detailed spectroscopic studies
described here probe the heme binding properties of apoNEAT-A and apo-NEAT-C. We report that the absorption and MCD data for NEAT-A, presented in Figs. 3 and 4, are indistinguishable from those of the whole IsdA protein [7], which offers strong support for use of the NEAT domain to study binding properties of the Isd proteins. In previous studies of IsdC, the optical spectra were dominated by iron-free PPIX, with only a minor heme component present [8,9]. This raised the question as to why IsdC preferentially bound PPIX even though it contained a heme binding NEAT domain. The heme binding environment in NEAT-C is different from that in NEATA, and the results presented in Figs. 5–8 show that heme does bind efficiently to apo-NEAT-C. Comparison of the spectroscopic data for NEAT-A and NEAT-C allow conclusions about the heme binding in the two proteins. First, we will consider the MS data. NEAT-A and NEAT-C exhibit very similar charge state spectra, in which the major charge states are dominated by a single species at +8, reminiscent of the data for myoglobin [17]. This pattern can be interpreted as arising from a globular structure. The data for NEAT-C are quite similar to the data for the entire IsdC protein, most likely because the NEAT domain represents 75% of the entire protein. The MS data we previously reported for IsdA, exhibited a broad distribution of peaks, which indicates the presence of a more extended conformation. However, NEAT-A comprises only 45% of the entire protein. We turn now to the optical spectra of the heme-boundNEAT domains. The data for NEAT-A were indistinguishable from the data of IsdA [7]. Because the NEAT domains are so specific in their heme binding properties we suggest that the heme binding properties of NEAT-C will represent those of IsdC. The MCD spectral data allows comparison of the heme binding sites based on accessible oxidation, spin, and ligation states [14,18,19]. Protein-based axial ligands control the spin state and stabilize the oxidation state of the heme iron. In addition, the accessibility to the surrounding environment is also controlled by the protein heme binding pocket. The MCD spectra showed that both NEAT-A and NEAT-C bind a high-spin ferric heme coordinated by a phenolate axial ligand (Figs. 3 and 6) [7,12], consistent with the recent X-ray structural data [4,16]. Neither NEAT-A nor NEAT-C bind cyanide, to give the expected low-spin ferric heme, which indicates a restricted access to anionic ligands [14]. Reduction of the heme iron using sodium hydrosulfite results in contrasting results for NEAT-A and NEAT-C. In NEAT-A high-spin ferrous heme was formed first which bound strongly to added carbon monoxide which we interpret as indicating that the heme is bound to the distal histidine and a proximal CO [7,14]. However, when reduction of the heme in NEAT-C was carried out, only a fraction of the heme was initially reduced, even though excess reducing agent was used (Fig. 7A). Reduction of a greater fraction of heme took place following the addition of CO. The absorption and MCD spectra of the reduced and CO ligated heme in
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NEAT-C do not resemble those of NEAT-A or myoglobin, in which a histidine is present to bind ferrous heme. However, the NEAT-C spectra do resemble the spectroscopic studies of bis-carbonmonoxy-Fe(II)-heme in aqueous solution (Fig. 8). This result implies that the heme in NEAT-C is released following reduction. We can understand this behavior in terms of the absence of a histidine residue near the distal heme site, which is present in NEAT-A. Overall, the data reported here provide the first detailed spectral properties of heme-bound NEAT domains from S. aureus. It is interesting to note, however, that some NEAT domains do not readily bind heme and the reasons for these differences are under study. In the results presented here, we demonstrate that tyrosine as a proximal ligand to the iron stabilizes the ferric oxidation state. Further, while the heme is clearly accessible to reducing agents (we propose through the edge of the ring), the ferric iron is not accessible to even strong ligands such as cyanide. The presence of a histidine in the distal site of the heme pocket in NEAT-A is required to stabilize ferrous heme, so it is not surprising that NEAT-C, which lacks a histidine in the distal site, loses the heme following reduction. Following from these studies, we will continue to explore the heme binding chemistry of other NEAT domains to determine how general our findings are and to address questions such as (i) Does the absence of the histidine always result in weak binding for ferrous heme? and (ii) Is bound ferric heme always inaccessible to further axial ligation? If, as we currently understand, the NEAT domains are a key factor in heme-trafficking in Gram-positive bacteria, then elucidation of the heme binding properties of NEAT domains, alone and in combination, will aid in our understanding of the overall mechanism of heme acquisition.
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resuspended in binding buffer (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 10 mM imidazole), and ruptured in a French pressure cell. Insoluble material was removed by centrifugation at 150,000g for 1 h. Proteins were purified by passage of the cell lysate across a 5 mL HiTrap chelating column (GE Healthcare), and eluted from the column with elution buffer (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 500 mM imidazole). Finally, the isolated proteins were dialyzed against 20 mM Na-phosphate, pH 7.4, with 150 mM NaCl. 4.2. Heme loading Purified apo-NEAT-A and apo-NEAT-C were concentrated to approximately 150 lM. Protein concentrations were estimated using the extinction coefficients of 15,930 L M 1 cm 1 for apo-NEAT-A and 18,490 L M 1 cm 1 for apo-NEAT-C at 280 nm. Hemin (Fluka) in DMSO (1.9 mM) was added to 4 mL of protein in 1.5 excess. Heme concentrations were calculated using the pyridine hemochromogen test [6]. Following incubation for 2 h, protein samples were separated using G-25 size-exclusion chromatography. 4.3. Mass spectrometry Electrospray mass spectra were recorded on a Micromass LCT, time of flight mass spectrometer operating in the positive ion mode. Stock protein solutions (150 lM) were placed on a G-25 size-exclusion column and eluted with 20 mM ammonium formate buffer (pH 7.3). Protein fractions were collected and run within 30 min on the mass spectrometer. 4.4. Absorption and MCD spectroscopy
4. Experimental 4.1. NEAT domain overexpression DNA encoded NEAT-A and NEAT-C were PCRamplified from the S. aureus chromosome and cloned into pET28a(+) such that the expressed protein would incorporate an N-terminal 6His-tag. In the case of NEAT-A, the recombinant protein included residues 62–184 of the 350 amino acid long protein and for NEAT-C, residues 28– 150 of the 227 amino acid long protein (cell surfaceexpressed proteins in S. aureus are cleaved at both the N and C termini). The resulting recombinant plasmids were then digested with XbaI and HindIII, and the NEATencoding fragments cloned into pBAD30. Plasmids were then introduced into E. coli RP523 [5], a hemB mutant strain. Overexpression of His-tagged apo-NEAT domains in E. coli RP523 was achieved by growing plasmid-containing cultures in Luria–Bertani (LB) broth (Difco) with ampicillin (100 lg/mL) and L-arabinose (0.1%) at 30 °C overnight to induce protein expression. Cells were then recovered and
Absorption spectra (Cary 500, Varian Inc. Canada) and MCD spectra using an SM2 5.5 T superconducting magnet (Oxford Instruments Ltd., Oxford, UK) aligned in a J-820 CD spectrometer (Jasco Inc., Japan) were measured at room temperature. Heme-bound protein solutions were diluted to absorbances of less than 0.8 in the Soret region. Small amounts of crystalline sodium cyanide were added in approximately 5-fold, 10-fold and a large excess. In all cases, the absorption and MCD spectra in the UV–visible regions of the NEAT-A and NEAT-C proteins were unchanged following additions of the cyanide. To obtain the ferrous heme spectra, small amounts of crystalline sodium hydrosulfite were added until no further changes were observed in the absorption spectrum. Carbon monoxide was then bubbled into the reduced protein samples until no further changes were observed in the absorption spectra. Absorption spectra were recorded before and after MCD measurements to ensure re-oxidation of the heme did not occur. In all figures showing absorption and MCD data, the ordinate (y-axis) is scaled as DAL-R/M – that is the MCD signal intensity is in units of Tesla 1.
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The ordinate for the absorption spectrum is also absorbance so that both spectra scale linearly. The absorption and MCD spectra were recorded for the same solution at the time the MCD spectrum was measured. 5. Abbreviations ISD iron-regulated surface determinant CD circular dichroism MCD magnetic circular dichroism ESI-MS electrospray ionization mass spectrometry MRSA methicillin-resistant Staphylococcus aureus NEAT near iron transporter NEAT-A IsdA NEAT domain NEAT-C IsdC NEAT domain S. aureus Staphylococcus aureus
Acknowledgments D.E.H. is a member of the Infectious Diseases Research Group at UWO. We acknowledge financial support from the Natural Sciences and Engineering Research Council (to D.E.H. and M.J.S. (operating grants) and M.C.P. (student scholarship)), and technical support from Johnson Cheung. References [1] J.J. Bullen, E. Griffiths, Iron and Infection: Molecular, Physiological and Clinical Aspects, second ed., John Wiley and Sons, New York, 1999.
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Inorganic Biochemistry Journal of Inorganic Biochemistry 102 (2008) 480–488 www.elsevier.com/locate/jinorgbio
Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus q Mark Pluym a, Naomi Muryoi b, David E. Heinrichs b,*, Martin J. Stillman a,* b
a Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 15 October 2007; received in revised form 14 November 2007; accepted 15 November 2007 Available online 3 December 2007
Abstract Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Iron regulated surface determinant; Near iron transporter; Gram-positive bacteria; Iron acquisition; Magnetic circular dichroism spectroscopy; Heme binding protein; Electrospray ionization mass spectrometry; Heme iron spin states
1. Introduction Worldwide, Staphylococcus aureus (S. aureus) is one of the most important causes of life-threatening bacterial infections. It causes a variety of diseases ranging from mild skin and soft tissue infections to severe life-threatening infections. The rapid spread of multi-drug resistant strains, q This work was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to both D.E.H. and M.J.S.), and the NSERC Postgraduate Scholarship program (funding for M.P.). We acknowledge technical support provided by Johnson Cheung. * Corresponding authors. Tel.: +1 519 661 3984; fax: +1 519 661 3499 (D.E. Heinrichs), tel.: +1 519 661 3821; fax: +1 519 661 3022 (M.J. Stillman). E-mail addresses: [email protected] (D.E. Heinrichs), martin.stillman@ uwo.ca (M.J. Stillman).
0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.11.011
including methicillin-resistant S. aureus (MRSA), has added urgency to the development of effective treatment and prevention in those more susceptible to infection. Iron is an essential nutrient for almost all organisms, including most bacteria, but it is frequently a growth limiting nutrient due to its insolubility in the presence of oxygen and, in vivo, its sequestration by proteins both inside and outside of cells. Successful pathogens, including S. aureus, have developed mechanisms to thrive under the extreme levels of iron restriction present in the host environment. Heme iron, present within hemoglobin in red blood cells, or in myoglobin, represents the largest iron reservoir in the human body and can be a significant source of iron for some bacterial pathogens [1]. Recent studies have shown that S. aureus scavenges heme using iron-regulated surface determinant (Isd) proteins [2,3], of which four proteins, IsdA, IsdB, IsdC and
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HarA (or IsdH), are covalently anchored to the bacterial cell wall. Each possesses at least one copy of an approximately 150 amino acid residue domain referred to as a NEAT domain [2]. The widespread occurrence of the NEAT domain has led to a number of studies aimed at determining its specific function in proteins located at the Gram-positive bacterial cell surface [4–6,3]. In combination, and in agreement with previous spectroscopic studies [4–6,3], analysis of the crystal structures of S. aureus IsdA NEAT (NEAT-A) [3] and IsdC NEAT (NEAT-C) [4], have demonstrated that these particular domains coordinate one heme molecule with axial coordination of heme iron mediated by a single tyrosine residue. Based on the presence or absence of this tyrosine as well as other ‘heme-pocket’ residues [3], it is possible to subdivide NEAT domains into those predicted to bind heme and others that would not coordinate heme, at least in similar fashion. Given this, each of the four S. aureus cell-wall-anchored Isd proteins (i.e. IsdA, IsdB, IsdC and HarA/IsdH) contain one NEAT domain that binds heme. In this paper, we report studies using a combination of absorption and MCD spectroscopy and ESI-MS to elucidate details of the heme environment in the NEAT domains of IsdA (NEAT-A) and IsdC (NEAT-C). The absorption and MCD data indicate that both NEAT-A and NEAT-C bind high-spin ferric hemes. The heme binding pocket is inaccessible to small anionic ligands but reduction of the heme in NEAT-A opens access, allowing for CO axial ligation. In contrast, reduction of the heme in NEAT-C results in loss of the heme from the binding site of the protein. A comprehensive understanding of NEATdependent heme binding will aid in our ability to understand the mechanisms of heme binding and release, and possible heme transfer reactions between Isd proteins. 2. Results The NEAT domains, which are studied here are part of the sequences in the Isd cell-wall-anchored IsdA and IsdC proteins, and have been shown to function as the heme binding domains in a wide range of other heme binding proteins [4,3,7]. While spectroscopic data have been reported by us previously for IsdA [7], IsdC [8,9], and IsdE [9], there have been no MCD spectral data reported previously for isolated NEAT domains from any protein. In this study, the recombinant NEAT domains of S. aureus IsdA (NEAT-A) and IsdC (NEAT-C) proteins were used to further investigate the heme binding properties of the complete proteins, IsdA and IsdC. These studies also answer the question about how independently the NEAT domains act in binding heme in the Isd series of proteins. Previous studies have shown that IsdA efficiently scavenges heme from within Escherichia coli [7], whereas IsdC scavenges primarily PPIX [8,9]. For both proteins, the major fraction isolated following purification is the heme- or PPIX-bound holo form. Previous attempts at removing bound heme to form the apo-protein using the traditional 2-butanone
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extraction were unsuccessful because the remaining apoprotein aggregated and precipitated. However, utilizing heme-deficient E. coli proved an effective means of obtaining apo-NEAT-A and apo-NEAT-C. These species were then reconstituted with heme, which allowed for a complete spectroscopic study of the oxidation state and spin states of the heme iron bound in the NEAT-C and NEAT-A domains, as previously reported for IsdA [7]. The spectral data reported here also show that reconstituted NEAT-A exhibits heme binding characteristics almost identical to those of the whole IsdA protein, that is the heme-boundIsdA. We, therefore, infer that the heme binding properties of the NEAT-C species also represent those of the whole IsdC protein, of which there exist no extensive spectroscopic data [8]. 2.1. IsdA NEAT heme binding The mass spectrum of apo-NEAT-A, following purification from E. coli, shows a peak at 16,377 Da (Fig. 1B) that is associated with the heme-free or apo-NEAT-A protein. The relative intensities of the heme-free (16,377 Da) and hemebound (16,993 Da) peaks in Fig. 1B show that less than 10% of the protein is bound to heme. Two distributions of charge states exist in apo-NEAT-A, centered about +15 and +8 (Fig. 1A). Following the addition of hemin and purification using size-exclusion chromatography, these charge states remain unchanged, with the two distributions centered as before at +15 and +8 (Fig. 1C). Following this heme loading reaction, over 95% of the protein is observed to be bound to heme, as shown by the intensification of the peak at 16,995 Da (Fig. 1D). Furthermore only trace amounts of free heme are present (616 m/z in Fig. 1C). The fact that the observed charge states remain unchanged following heme binding suggests that little conformation change occurs as a result of heme binding [10,11]. This is in complete agreement with crystal structure data for apo and holo NEAT-A that was recently reported [3]. As described above, a small amount of heme was bound to NEAT-A following purification of the predominantly apo-NEAT-A from E. coli, as shown in Fig. 1B. The corresponding absorption spectrum is shown in Fig. 2A, which exhibits the characteristic bands of a high-spin ferric heme, in particular the charge transfer band at 628 nm [7,12,13]. The strong intensity of the 277 nm protein band in relation to the Soret band indicates the relative abundance of apoNEAT-A as observed in the mass spectrum. As expected, following heme loading the Soret band becomes much more intense in relation to the 276 nm protein band. The absorption and MCD data presented in Fig. 3A show that like whole IsdA protein, the iron in heme-bound-NEAT-A is a 5-coordinate, high-spin, Fe(III) heme, which, based on our previous MCD studies and the results of X-ray diffraction analysis, is known to be coordinated by a tyrosine residue [7,12]. Axial ligation by cyanide in the ferric heme state is restricted (Fig. 2B) as reported for the whole IsdA protein. However, following reduction
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Fig. 2. Absorption spectra of NEAT-A (A) following purification from E. coli and (B) following the addition of hemin.
2.2. IsdC NEAT heme binding
Fig. 1. Charge state and mass spectra of apo-NEAT-A and holo-NEATA. (A) Charge state spectrum of NEAT-A following purification from E. coli. (B) Deconvoluted mass spectrum of NEAT-A showing apoNEAT-A (16,377 Da) and holo-NEAT-A (16,993 Da). (C) Charge state spectrum of NEAT-A following the addition of hemin. (D) Deconvoluted mass spectrum of NEAT-A following the addition of hemin showing apoNEAT-A (16,379 Da) and holo-NEAT-A (16,995 Da).
(Fig. 4A) access was possible for carbon monoxide, which binds strongly to the Fe(II), dramatically changing the optical spectra (Fig. 4C) [7,14]. From this we can conclude that the protein regions outside of the NEAT domain of IsdA play little role in heme binding, most likely acting as a scaffold for the NEAT domain. The data presented here indicate that NEAT-A has ligand binding properties like IsdA.
Previous reports on the heme binding properties of IsdC were complicated due to the predominance of PPIX following isolation from E. coli [8]. While a small fraction of heme was bound to the IsdC as isolated, the optical spectra were dominated by the presence of the PPIX. In this work, we now can report the heme binding properties of apoNEAT-C following reconstitution with hemin. The presence of the peak at 16,192 Da in the mass spectral data shown in Fig. 5 clearly show that following purification the majority (95%) of the NEAT-C produced recombinantly is heme-free (Fig. 5B). Significantly, there is no indication that free PPIX is in solution, because no band at 564 m/z is observed [8]. Following hemin loading, approximately 95% of the protein is heme-bound (16,809 Da in Fig. 5D). The charge state spectrum of apo-NEAT-C is dominated by the +8 and +9 charge states (Fig. 5A). This distribution is unchanged following heme loading (Fig. 5C) suggesting little conformation change in the protein takes place after heme binding, a result similar to the data for NEAT-A [10,11]. Again, the lack of change in the charge states points to the value of mass spectral data in determining conformational changes upon substrate binding. As described above from the ESI-MS data, the apoNEAT-C sample included a small fraction bound to heme
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Fig. 3. Absorption and MCD spectra of ferric heme loaded NEAT-A. (A and B) Following purification and (C and D) following the addition of sodium cyanide. In this and all figures showing absorption and MCD data, the ordinate (y-axis) is scaled as DAL-R/M; that is, the MCD signal intensity is in units of Tesla 1. The ordinate for the absorption spectrum is also absorbance so that both spectra scale linearly. The absorption and MCD spectra were recorded for the same solution at the time the MCD spectrum was measured.
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Fig. 4. Absorption and MCD spectra of ferrous heme loaded NEAT-A. (A and B) Following the addition of sodium hydrosulfite and (C and D) following the addition of sodium hydrosulfite and carbon monoxide.
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Fig. 5. Charge state and mass spectra of apo-NEAT-C and holo-NEATC. (A) Charge state spectrum of NEAT-C following purification from E. coli. (B) Deconvoluted mass spectrum of NEAT-C showing apoNEAT-C (16,192 Da) and holo-NEAT-C (16,808 Da). (C) Charge state spectrum of NEAT-C following the addition of hemin. (D) Deconvoluted mass spectrum of NEAT-C following the addition of hemin showing apoNEAT-C (16,193 Da) and holo-NEAT-C (16,809 Da).
following purification of the isolate from E. coli. This small component exhibited an absorption spectrum with a Soret band at 404 nm and visible region bands at 501, 534, and 625 nm. Following heme loading and subsequent purification, a matching, but far more intense, spectrum was obtained in which the Soret band absorbance now greatly
Fig. 6. Absorption and MCD spectra of ferric heme loaded NEAT-C. (A and B) Following chromatographic separation and (C and D) following the addition of sodium cyanide.
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Fig. 7. Absorption and MCD spectra of ferrous heme loaded NEAT-C. (A and B) Following the addition of sodium hydrosulfite and (C and D) following the addition of sodium hydrosulfite and carbon monoxide.
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exceeds that of the NEAT-C residues in the 277 nm region. This result is good evidence that the heme added to the apo-NEAT-C binds in the same manner as the heme that bound in the E. coli. The MCD spectrum of NEAT-C, following the addition of excess heme and purification, is shown in Fig. 6B. This detailed spectrum is very similar to that recorded for NEAT-A. From our analysis of the absorption and MCD spectra of NEAT-A we can, therefore, conclude that NEAT-C also binds a 5-coordinate, high-spin, ferric heme through a proximal tyrosine residue [4,8,12]. The test of access to the heme iron, using cyanide, again failed and did not result in a low-spin ferric iron center, from which we conclude that axial access to the heme iron is also blocked in NEAT-C. However, there were differences between NEAT-C and NEAT-A following reduction of the heme. The addition of sodium hydrosulfite to NEATC resulted in a very complex spectrum representative of a mixture of hemes (Fig. 7). This is quite unlike the case with NEAT-A, where reduction of the heme resulted in formation of a high-spin ferrous heme, coordinated by a histidine residue, as reported for IsdA [7,14]. Unlike NEAT-A, NEAT-C does not contain a histidine residue near the distal heme site; instead, an isoleucine is present [4,3]. The bands arising from the presence of both bound ferric heme and reduced ferrous heme produce the complex absorption and MCD spectra shown in Fig. 7A. It should be noted that an approximately 2 greater concentration of sodium hydrosulfite was required to reduce the heme in NEAT-C to obtain the spectrum in Fig. 7A than that required to reduce the heme in NEAT-A. The mixture of bands in the spectra, and the high levels of reducing agent required, suggest that in fact the ferrous heme is released by NEATC and the remaining bound heme is still oxidized. We confirmed this in experiments using CO. Following the addition of sodium hydrosulfite, and then carbon monoxide, to NEAT-C, an MCD spectrum different to that of NEAT-A was recorded. Instead of a 2:1 intensity ratio between the Soret and Q band A-terms, a ratio closer to 1:1.5 was observed. Furthermore, the band positions were blue-shifted. For NEAT-C, the A-term crossover points were observed at 410 and 565 nm, whereas for NEAT-A the A-term crossover points were observed at 421 and 569 nm. As we have noted previously, the spectral patterns for different oxidation, spin and ligand states possible for heme are surprisingly consistent across many proteins, so these changes must be the result of formation of a different species. The absorption and MCD spectra of hemin in a 1:1 mixture of DMSO and 1 PBS, and following the addition of sodium hydrosulfite and carbon monoxide, are shown in Fig. 8. The bands observed are very similar to those of NEAT-C following the same chemistry (Fig. 7B), with MCD crossover points at 410 and 563 nm. We propose then that following reduction, NEAT-C releases bound heme and upon addition of carbon monoxide, the heme becomes coordinated to CO.
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Fig. 8. Absorption (A) and MCD (B) spectra of bis-CO ferrous heme in a 1:1 mixture of DMSO and 1 PBS.
3. Discussion The bacterial iron-regulated Isd proteins were first identified in S. aureus [15] but are also present in other Gram-positive bacteria, such as Listeria monocytogenes and Bacillus anthracis. A subset of the Isd proteins, IsdA, IsdB, IsdC and IsdH, are proposed to take part in the extracytoplasmic membrane transfer of heme from host serum heme-proteins through to membrane-localized heme transporters. Our initial studies of the heme binding properties of IsdA [7], IsdC [8], and IsdE [9] were based on recombinant proteins representative of the full length proteins, as expressed in the cytoplasm of E. coli. Both IsdA and IsdC contain a single copy of the NEAT domain, which operates as the heme binding site [4,16]. Studying the isolated NEAT domains of IsdA and IsdC has several advantages over studying the entire proteins. Most significantly, the increased stability observed for the NEAT domains in buffered solution allows for greater storage periods without any apparent aggregation or degradation and allows more detailed studies of the mechanistic properties of these proteins. Focusing strictly on the NEAT domains of the proteins, however, assumes that the removed portions of the proteins (see Experimental) have no or insignificant effect on the heme binding properties of the holo-proteins. The detailed spectroscopic studies
described here probe the heme binding properties of apoNEAT-A and apo-NEAT-C. We report that the absorption and MCD data for NEAT-A, presented in Figs. 3 and 4, are indistinguishable from those of the whole IsdA protein [7], which offers strong support for use of the NEAT domain to study binding properties of the Isd proteins. In previous studies of IsdC, the optical spectra were dominated by iron-free PPIX, with only a minor heme component present [8,9]. This raised the question as to why IsdC preferentially bound PPIX even though it contained a heme binding NEAT domain. The heme binding environment in NEAT-C is different from that in NEATA, and the results presented in Figs. 5–8 show that heme does bind efficiently to apo-NEAT-C. Comparison of the spectroscopic data for NEAT-A and NEAT-C allow conclusions about the heme binding in the two proteins. First, we will consider the MS data. NEAT-A and NEAT-C exhibit very similar charge state spectra, in which the major charge states are dominated by a single species at +8, reminiscent of the data for myoglobin [17]. This pattern can be interpreted as arising from a globular structure. The data for NEAT-C are quite similar to the data for the entire IsdC protein, most likely because the NEAT domain represents 75% of the entire protein. The MS data we previously reported for IsdA, exhibited a broad distribution of peaks, which indicates the presence of a more extended conformation. However, NEAT-A comprises only 45% of the entire protein. We turn now to the optical spectra of the heme-boundNEAT domains. The data for NEAT-A were indistinguishable from the data of IsdA [7]. Because the NEAT domains are so specific in their heme binding properties we suggest that the heme binding properties of NEAT-C will represent those of IsdC. The MCD spectral data allows comparison of the heme binding sites based on accessible oxidation, spin, and ligation states [14,18,19]. Protein-based axial ligands control the spin state and stabilize the oxidation state of the heme iron. In addition, the accessibility to the surrounding environment is also controlled by the protein heme binding pocket. The MCD spectra showed that both NEAT-A and NEAT-C bind a high-spin ferric heme coordinated by a phenolate axial ligand (Figs. 3 and 6) [7,12], consistent with the recent X-ray structural data [4,16]. Neither NEAT-A nor NEAT-C bind cyanide, to give the expected low-spin ferric heme, which indicates a restricted access to anionic ligands [14]. Reduction of the heme iron using sodium hydrosulfite results in contrasting results for NEAT-A and NEAT-C. In NEAT-A high-spin ferrous heme was formed first which bound strongly to added carbon monoxide which we interpret as indicating that the heme is bound to the distal histidine and a proximal CO [7,14]. However, when reduction of the heme in NEAT-C was carried out, only a fraction of the heme was initially reduced, even though excess reducing agent was used (Fig. 7A). Reduction of a greater fraction of heme took place following the addition of CO. The absorption and MCD spectra of the reduced and CO ligated heme in
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NEAT-C do not resemble those of NEAT-A or myoglobin, in which a histidine is present to bind ferrous heme. However, the NEAT-C spectra do resemble the spectroscopic studies of bis-carbonmonoxy-Fe(II)-heme in aqueous solution (Fig. 8). This result implies that the heme in NEAT-C is released following reduction. We can understand this behavior in terms of the absence of a histidine residue near the distal heme site, which is present in NEAT-A. Overall, the data reported here provide the first detailed spectral properties of heme-bound NEAT domains from S. aureus. It is interesting to note, however, that some NEAT domains do not readily bind heme and the reasons for these differences are under study. In the results presented here, we demonstrate that tyrosine as a proximal ligand to the iron stabilizes the ferric oxidation state. Further, while the heme is clearly accessible to reducing agents (we propose through the edge of the ring), the ferric iron is not accessible to even strong ligands such as cyanide. The presence of a histidine in the distal site of the heme pocket in NEAT-A is required to stabilize ferrous heme, so it is not surprising that NEAT-C, which lacks a histidine in the distal site, loses the heme following reduction. Following from these studies, we will continue to explore the heme binding chemistry of other NEAT domains to determine how general our findings are and to address questions such as (i) Does the absence of the histidine always result in weak binding for ferrous heme? and (ii) Is bound ferric heme always inaccessible to further axial ligation? If, as we currently understand, the NEAT domains are a key factor in heme-trafficking in Gram-positive bacteria, then elucidation of the heme binding properties of NEAT domains, alone and in combination, will aid in our understanding of the overall mechanism of heme acquisition.
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resuspended in binding buffer (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 10 mM imidazole), and ruptured in a French pressure cell. Insoluble material was removed by centrifugation at 150,000g for 1 h. Proteins were purified by passage of the cell lysate across a 5 mL HiTrap chelating column (GE Healthcare), and eluted from the column with elution buffer (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 500 mM imidazole). Finally, the isolated proteins were dialyzed against 20 mM Na-phosphate, pH 7.4, with 150 mM NaCl. 4.2. Heme loading Purified apo-NEAT-A and apo-NEAT-C were concentrated to approximately 150 lM. Protein concentrations were estimated using the extinction coefficients of 15,930 L M 1 cm 1 for apo-NEAT-A and 18,490 L M 1 cm 1 for apo-NEAT-C at 280 nm. Hemin (Fluka) in DMSO (1.9 mM) was added to 4 mL of protein in 1.5 excess. Heme concentrations were calculated using the pyridine hemochromogen test [6]. Following incubation for 2 h, protein samples were separated using G-25 size-exclusion chromatography. 4.3. Mass spectrometry Electrospray mass spectra were recorded on a Micromass LCT, time of flight mass spectrometer operating in the positive ion mode. Stock protein solutions (150 lM) were placed on a G-25 size-exclusion column and eluted with 20 mM ammonium formate buffer (pH 7.3). Protein fractions were collected and run within 30 min on the mass spectrometer. 4.4. Absorption and MCD spectroscopy
4. Experimental 4.1. NEAT domain overexpression DNA encoded NEAT-A and NEAT-C were PCRamplified from the S. aureus chromosome and cloned into pET28a(+) such that the expressed protein would incorporate an N-terminal 6His-tag. In the case of NEAT-A, the recombinant protein included residues 62–184 of the 350 amino acid long protein and for NEAT-C, residues 28– 150 of the 227 amino acid long protein (cell surfaceexpressed proteins in S. aureus are cleaved at both the N and C termini). The resulting recombinant plasmids were then digested with XbaI and HindIII, and the NEATencoding fragments cloned into pBAD30. Plasmids were then introduced into E. coli RP523 [5], a hemB mutant strain. Overexpression of His-tagged apo-NEAT domains in E. coli RP523 was achieved by growing plasmid-containing cultures in Luria–Bertani (LB) broth (Difco) with ampicillin (100 lg/mL) and L-arabinose (0.1%) at 30 °C overnight to induce protein expression. Cells were then recovered and
Absorption spectra (Cary 500, Varian Inc. Canada) and MCD spectra using an SM2 5.5 T superconducting magnet (Oxford Instruments Ltd., Oxford, UK) aligned in a J-820 CD spectrometer (Jasco Inc., Japan) were measured at room temperature. Heme-bound protein solutions were diluted to absorbances of less than 0.8 in the Soret region. Small amounts of crystalline sodium cyanide were added in approximately 5-fold, 10-fold and a large excess. In all cases, the absorption and MCD spectra in the UV–visible regions of the NEAT-A and NEAT-C proteins were unchanged following additions of the cyanide. To obtain the ferrous heme spectra, small amounts of crystalline sodium hydrosulfite were added until no further changes were observed in the absorption spectrum. Carbon monoxide was then bubbled into the reduced protein samples until no further changes were observed in the absorption spectra. Absorption spectra were recorded before and after MCD measurements to ensure re-oxidation of the heme did not occur. In all figures showing absorption and MCD data, the ordinate (y-axis) is scaled as DAL-R/M – that is the MCD signal intensity is in units of Tesla 1.
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The ordinate for the absorption spectrum is also absorbance so that both spectra scale linearly. The absorption and MCD spectra were recorded for the same solution at the time the MCD spectrum was measured. 5. Abbreviations ISD iron-regulated surface determinant CD circular dichroism MCD magnetic circular dichroism ESI-MS electrospray ionization mass spectrometry MRSA methicillin-resistant Staphylococcus aureus NEAT near iron transporter NEAT-A IsdA NEAT domain NEAT-C IsdC NEAT domain S. aureus Staphylococcus aureus
Acknowledgments D.E.H. is a member of the Infectious Diseases Research Group at UWO. We acknowledge financial support from the Natural Sciences and Engineering Research Council (to D.E.H. and M.J.S. (operating grants) and M.C.P. (student scholarship)), and technical support from Johnson Cheung. References [1] J.J. Bullen, E. Griffiths, Iron and Infection: Molecular, Physiological and Clinical Aspects, second ed., John Wiley and Sons, New York, 1999.
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