MhuD from Mycobacterium tuberculosis - probing a dual role in heme storage and degradation

Sarah J. Matthews1, Kamila J. Pacholarz1, Aidan P. France1, Thomas A. Jowitt2, Sam Hay1, Perdita E. Barran1, Andrew W. Munro1*

1Manchester Institute of Biotechnology, The University of Manchester, School of Chemistry, Faculty of Science and Engineering, 131 Princess Street, Manchester M1 7DN, UK. 2The Biomolecular Analysis Facility, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, Manchester M13 9PT, UK.

*Author for Correspondence: Prof. Andrew W. Munro. Email: Andrew.Munro@Manchester.ac.uk; Phone 0161 3065151.

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD diheme dissociation is far slower than monoheme dissociation at ~0.12 min-1 and ~0.25 s-1, respectively, indicating that MhuD has strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

KEYWORDS: Mycobacterium tuberculosis, heme oxygenase, heme degradation, heme storage, iron acquisition, native mass spectrometry

Heme degradation is an important process that is catalyzed by heme oxygenase enzymes. Canonical heme oxygenases catabolize heme through consumption of three oxygen molecules and seven reducing equivalents; this releases free iron, a molecule of carbon monoxide, and the linear tetrapyrrole biliverdin 1. The process is crucial for iron recycling, and is important for mammals in aspects such as antioxidant defense 2,3 and cell signalling 4,5. In mammals, reducing equivalents are supplied to heme oxygenases by NADPH-cytochrome P450 reductase 6, whereas ferredoxin is utilized in photosynthetic organisms that require biliverdin for synthesis of light-harvesting pigments 7. In several bacterial strains, including Deinococcus radiodurans and Pseudomonas aeruginosa, biliverdin also functions as a precursor for chromophores that function in bacteriophytochromes 8,9.

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Heme oxygenases also serve an important function in pathogenic bacteria, where iron acquisition is vital for successful growth, survival and pathogenicity. Adult humans have between 3–5 g iron distributed throughout the body 10. However, very little iron is freely available due to its insolubility 11 and its tendency to cause oxidative stress through H2O2-mediated free-radical generation 12,13. Most of the body’s iron is complexed in heme, where it is retained within hemoglobin, myoglobin, and cytochromes (~67%, ~3.5%, and ~3% of total body iron, respectively) 14.

Pathogenic bacteria have evolved mechanisms that allow them to scavenge iron from the host, with several of these strategies targeted towards uptake of heme. Once acquired and shuttled into the bacterial cytoplasm, heme can either be directly incorporated into bacterial proteins, or degraded by heme oxygenases 13,15. Many bacterial heme oxygenases are members of a group of canonical heme oxygenases that include HmuO from Corynebacterium diphtheria 16 and HemO from Neisseria meningitidis 17. Similarly to their mammalian counterparts, these enzymes degrade heme to biliverdin, and display an α-only structure that typically contains nine to ten helices 13. Another group, known as the IsdG family of heme oxygenases, have been identified in several organisms including Staphylococcus aureus, Bacillus anthracis and Listeria monocytogenes 18,19. The IsdG family of heme oxygenases was first discovered in S. aureus, which contains the two paralogous heme oxygenases IsdG and IsdI 18. Unlike the canonical heme oxygenases, these proteins are homodimers with a β-barrel conformation at the dimer interface, formed by monomers with a ferredoxin-like α/β-sandwich motif 20,21. In addition to differing structures of IsdG-type enzymes, compared with canonical heme oxygenases, the active site chemistry of the IsdG-type proteins precludes conversion of alternative linear tetrapyrroles to biliverdin 22. For example, it has been shown that, in IsdG and IsdI, degradation of heme produces free iron, a molecule of formaldehyde and the linear tetrapyrrole staphylobilin 20,23.

MhuD is an IsdG-type heme oxygenase from Mycobacterium tuberculosis (Mtb) 24. Tuberculosis (TB) is a global epidemic that in 2016 was responsible for over 1.6 million deaths worldwide. Increasing incidences of drug-resistant forms of TB are also concerning, with 600,000 new cases of rifampicin-resistant and 490,000 of multidrug-resistant TB reported in 2016 25. Hence, it is important to research new targets for treating TB, and MhuD has been suggested as one such potential target 26.

MhuD is upregulated 2.5-fold during macrophage infection, suggesting that this enzyme plays an important role in the pathogenicity of Mtb 27. Interestingly, crystallographic and isothermal titration calorimetry data have indicated that MhuD is able to bind two molecules of heme within each monomer, and that in this diheme state the enzyme is inactive (see Figure 1A) 24. This means that one dimeric heme protein is capable of binding up to four heme molecules, a facet that is apparently unique to MhuD among the heme oxygenases.

In the monoheme form, MhuD can oxidatively degrade heme to generate free iron and the unique tetrapyrrole mycobilin without the release of any additional small molecules 28,29. Mechanistic interpretation has been aided by crystal structures of the diheme- and monoheme-CN MhuD complexes 24,28. In the monoheme-CN MhuD structure, heme iron is ligated to the α2 helix through a His75 imidazole, and the α2 helix is kinked after residue Asn68 (see Figure 1B). In the diheme structure the α2 helix is extended, which causes a movement of the helix and the subsequent loop region away from the protein core, thus providing extra space for the binding of a second heme molecule. This His75-ligated heme molecule (solvent-exposed heme) is stabilized through several

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hydrophobic interactions, and by hydrogen bonding between the heme propionate groups and active site amino acids. Asn7 is positioned distally to the bound heme and is thought to be important for catalysis. The monoheme, cyanide-bound MhuD structure shows that a cyanide molecule is hydrogen bonded to Asn7 28, and N7A mutagenesis in IsdG abolishes heme degrading activity 21. In the diheme-MhuD structure, the solvent-protected heme is coordinated to the Asn7 residue via interactions with a chloride ion 24. Other important residues in the active site of MhuD include Trp66, where steric clashes with the heme substrate induce ruffling in the heme and allow the reaction mechanism to occur 30. Two arginine residues (Arg22 and Arg26) also play an important role in stabilizing the heme propionates and, when mutated, degradation of heme forms biliverdin instead of the natural mycobilin product 31.

The ability of MhuD to degrade heme in the monoheme form is thought to be important for providing an iron source for Mtb during iron-depleted conditions. The formation of diheme-MhuD is intriguing, and current hypotheses suggest this form functions in heme storage. Thus, MhuD is able to provide evolutionarily effective mechanisms for both heme storage and degradation in the same scaffold 24. In this study, we have investigated the stoichiometry and distributions of MhuD-heme binding in detail using native mass spectrometry and activated ion mobility mass spectrometry (aIM-MS) approaches 32,33.

Results and Discussion

Native Mass SpectrometryThe purification of MhuD was performed by immobilization of MhuD on a Ni-NTA column, followed by proteolytic cleavage of the polyhistidine tag from MhuD to facilitate elution of high purity apo-MhuD (see Figure S1 and Figure 2).

Nanoelectrospray ionization-mass spectrometry (nESI-MS) revealed that MhuD is dimeric in solution, with very little monomeric protein present. The mass spectrum displayed three charge states from m/z 2513 to m/z 3230, corresponding to dimeric MhuD [M + 9H]9+, [M + 8H]8+ and [M + 7H]7+, respectively (Figure 2). The spectrum was deconvoluted using UniDec software (http://unidec.chem.ox.ac.uk/) to reveal a single protein species with an average mass of 22620 Da, which corresponds to dimeric apo-MhuD with a theoretical average mass of 22598 Da. The minor difference between the theoretical and experimental mass arises from the presence of residual solvent not completely stripped during the desolvation process.

For clarity, further discussions of the MhuD protein will refer to the enzyme in its dimeric form. To study the stoichiometry of heme binding to MhuD, 5 µM MhuD was incubated overnight at 4°C with the following heme concentrations before analysis by nESI-MS: 0, 2.5, 5, 10, 20, 30, 40, 60, 80 µM.

It has previously been hypothesized that, at low heme concentrations, single molecules of heme would bind in the MhuD active sites and would be degraded to release free iron. At high heme concentrations, MhuD would instead become inactive through binding of two heme molecules in a single monomer, thus storing up to 4 heme molecules. In this model, the Kd for binding of monoheme would be substantially tighter than that for the binding of a second heme to the monoheme to yield the diheme form. If this hypothesis were true we would see that, at lower heme

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concentrations, MhuD would only be present in apo-, 1-, or 2-heme bound forms; and 3- and 4-heme bound MhuD forms would only be formed after the complete conversion of apo- and 1-heme bound forms to 2-heme bound MhuD (Figure 2 and Figure S3).

As would be expected, the addition of increasing concentrations of heme to 5 µM MhuD results in decreasing levels of apo-MhuD, and increasing levels of the heme-bound forms of MhuD, with deconvoluted masses of 23230, 23870, 24480 and 25115 Da, corresponding to the 1-, 2-, 3-, and 4-heme bound forms of MhuD, respectively (Figure 3). Upon the addition of 2.5 µM heme, the apo-MhuD form remains the predominant species at around 55%, with the 1-, and 2-heme bound species formed in ~33% and 12% proportions, respectively.

Increasing the heme concentration to 5 µM further decreases the proportion of apo-heme MhuD to ~33%. Although the concentration of 1-heme MhuD remains relatively constant at 34%, the abundance of the 2-heme form of MhuD increases to ~23% and the emergence of a 3-heme species is observed (~10%). The development of a 3-heme species, whilst there is still a large proportion of apo-MhuD, contradicts the theory that the Kd for monoheme binding is much tighter than that for the subsequent binding of other hemes. Furthermore, the 4-heme bound form of MhuD becomes apparent at a heme concentration of 10 µM (at 8%) where apo- and 1-heme forms of MhuD are present at concentrations of ~14% and 25%, respectively. In fact, the apo-heme form is still present at heme concentrations up to 20 µM, which is four times the concentration of the MhuD.

By plotting the applied heme concentration against relative levels of MhuD-bound heme, data were fitted using the Morrison equation 34 with an adjusted R2 value of 0.996 and enzyme concentration of 18.4 ± 1.5 µM (in good agreement with the expected binding site concentration of ~20 µM) , giving an apparent Kd of 1.3 ± 0.7 µM. This suggests tight binding of heme to MhuD without significant cooperativity.

Data for individual species were modelled with a sequential binding mechanism (Eq 1; Figure 3A), which provided estimates for four separate Kd values of Kd,1 0.45 µM, Kd,2 0.55 µM, Kd,3 1.1 µM and Kd,4

2.2 µM. These Kd values describe a model where binding of the final heme (Kd,4) occurs with approximately 5-fold weaker affinity than binding of the first heme molecule (Kd,1). This is consistent with our findings that the emergence of a 3-heme bound MhuD occurs even when there is still apo-MhuD present. The values of Kd1 and Kd2 are not significantly different (vide infra), which suggests that the predominant 2-heme bound species is that with one heme bound to each monomer. Similarly the Kd3 and Kd4 are not significantly different (within error) and likely report on the binding of the second heme molecule to each binding site (see “Modelling of Kd values for MhuD-heme binding” section below). However, the Kd1 and Kd3 values are quite similar, and thus it is possible for 2-heme bound MhuD to contain 2-heme molecules in one monomer whilst the other is in the apo form. To further explore this phenomenon, we performed activated ion mobility experiments as discussed below.

The conformational sizes of the different MhuD forms were investigated using ion mobility MS (IM-MS). Collision cross sections measured with nitrogen as the buffer gas (TWCCSN2) were calculated for the [M + 9H]9+, [M + 8H]8+ and [M + 7H]7+ charge states for the apo, 1-, 2-, 3- and 4-heme bound forms of MhuD. Median TWCCSN2 values of 15.48 ± 0.21, 15.79 ± 0.30, 16.14 ± 0.21, 16.49 ± 0.13, and 16.77 ± 0.18 were determined for the apo, 1-, 2-, 3-, and 4-heme bound forms of MhuD (Figure S3 and Table 1). These values represent small, but consistent, increases in TWCCSN2 with increasing heme

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binding, concurrent with restructuring due to the opening of the α2 helix and its following loop region, with no large structural rearrangements.

To ensure that 3- and 4-heme bound MhuD were formed through sequential binding of heme molecules, and not by an artefact of dimeric heme molecule binding, we explored the binding of deuteroheme to MhuD using nESI-MS. Due to the absence of the vinyl groups present in heme, deuteroheme exhibits a weaker Kd for dimerization, and studies have shown that the Kd for dimerization is 1.87 x 10-5 M-1 for deuteroheme compared with 4.7 x 10-7 M-1 for protoheme 35.

5 µM MhuD was incubated with 10 µM deuteroheme overnight at 4°C prior to MS analysis. Compared with the heme b-binding spectra, the peaks in the deuteroheme spectra were broader and noisier, presumably due to an increased presence of salt adducts (Figure S5). Nevertheless, [M + 8H]8+ and [M + 7H]7+ charge states clearly showed the presence of 3- and 4-deuteroheme bound forms of MhuD in addition to apo-, 1-, and 2-heme bound species. These data indicate that diheme binding is a physiologically relevant event, and further supports the specific role for MhuD in heme storage.

To further explore the distribution of bound heme(s) within the dimeric MhuD, we employed activated ion mobility mass spectrometry (aIM-MS) to probe unfolding and heme dissociation of the 1-, 2-, 3- and 4-heme [M + 8H]8+ charge states by activating the protein ions via sequential ramping of the collision voltage prior to ion mobility analysis 33 (Figure 4).

Using a sample of 5 µM MhuD that had been incubated with 10 µM hemin, we first mass-selected the ion corresponding to the 1-heme bound MhuD [M + 8H]8+ with charge state (m/z 2904), and performed aIM-MS by increasing the voltage in the trap collision cell from 0–200 V. The 2D IM-MS plot shows two main protein unfolding events (at around 25 V and 50 V), where the second unfolding step gives rise to a purely apo-dimeric MhuD species, in addition to dissociation of dimeric MhuD into separate monomers (m/z 2261 corresponding to a [M + 8H]5+ charge state) (see Figure 4). When this experiment was repeated for the 2-heme bound form of MhuD [M + 8H]8+ with charge state (m/z 2982), we observed a similar unfolding profile to that for the 1-heme bound state. However, the abundance of monomeric-MhuD produced upon activation was decreased compared with the 1-heme bound MhuD species. Furthermore, activation of the 3- and 4-heme bound [M + 8H]8+ charge states (m/z 3060 and 3139) did not give rise to the monomeric MhuD species. We speculate that additional bound heme groups may stabilize the dimer interface, hence requiring more energy to induce dimer dissociation. Upon activation of the 3- and 4-heme bound forms of MhuD, the second unfolding step gave rise to MhuD complexed with a mixture of heme stoichiometries. This finding differs from the 1- and 2-heme bound forms of MhuD, where apo-MhuD was formed upon the second unfolding step. In the 3-heme bound MhuD form, a third unfolding step is observed, where all heme dissociates from MhuD to form the apo-dimeric MhuD [M + 8H]8+ charge state species. This event does not occur in the 4-heme bound MhuD, indicating that full heme occupation of MhuD alters the intrinsic properties of the protein.

The crystal structures of diheme-MhuD and heme-CN bound MhuD are very similar, with the most notable differences affecting the α2 helix, to which heme iron is ligated through the His75 residue, and the subsequent loop region 24,28. These aIM-MS data suggest that diheme binding may have implications for the properties of the overall dimeric complex and its propensity to become monomeric upon activation.

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Surprisingly, upon unfolding of the protein, the first heme species to be released and observed in the mass spectrum was m/z 1250, which corresponds to diheme (m/z 1232) complexed with a molecule of water (+ 18 Da) (Figure 5a and Figure S6). This suggests that the affinity for the heme dimer is greater than the affinity of heme for the His75 residue of MhuD. Further increasing the collision voltage then caused a mass loss of 18 corresponding to dissociation of a water molecule to produce diheme, and eventually dissociation of the diheme complex to the monoheme species (m/z 616). The same pattern is observed for 3-, and 4-heme bound forms. We cannot infer the proportions of monoheme and diheme forms being produced through activation of the protein species. However, these data are interesting as diheme dissociation from 2-heme bound MhuD suggests that MhuD can bind two molecules of heme in one of its monomers whilst the other monomer is in its apo form (Figure 5B). Therefore, it is not necessary for both subunits to be in the monoheme form prior to binding of a second heme molecule.

The diheme bound MhuD crystal structure 24 depicts a chloride ion ligating to and bridging the solvent-protected heme iron to the Asn7 residue. Hence, there may be a crucial role for a solvent-protected heme ligand in linking the heme to the Asn7 residue and positioning the heme within the active site. The aIM-MS plots show that the diheme species has a longer drift time compared with the diheme-water complex (Figure 5B). This indicates that, when diheme is complexed with water, the two heme molecules are brought into closer proximity. This could be important for heme storage, enhancing diheme binding affinity within MhuD.

Analytical ultracentrifugationAnalytical ultracentrifugation (AUC) was used to further probe the oligomeric state of MhuD. MhuD displayed a sedimentation coefficient of 2.08S, and a frictional ratio of 1.39 (Figure S2), which correlates with a protein mass of 22100 Da, and is consistent with the theoretical mass of dimeric MhuD (22598 Da). Dilution of the protein within detectable levels (0.5 μM dimeric-MhuD) resulted in no change to the sedimentation coefficient, and no appearance of a monomeric-MhuD species. These results support the nESI-MS findings that MhuD is a dimeric protein in solution. With reference to the dimeric MhuD crystal structure shown in Figure 1, it is likely that the interfacial beta-sheet interactions observed in the MhuD dimer structure are also those that stabilize MhuD in the solution state.

Apomyoglobin assaysDetermination of the kon value for heme binding is hindered by the propensity of heme to aggregate at micromolar concentrations in solution, which precludes analysis by stopped-flow kinetics. Instead we utilized the rapid kon (~1 × 108 M-1 s-1) 36 of apomyoglobin to measure the rate of dissociation of heme from MhuD, and its subsequent binding to apomyoglobin to form a distinct species. This allowed us to determine the rate of heme dissociation for MhuD, as previously performed with IsdG and IsdI 37.

To determine whether there was any difference between the rate of dissociation for the solvent-exposed and solvent-accessible hemes, heme was preincubated with different concentrations of MhuD to achieve a range of heme stoichiometries and distributions. The MhuD-heme complexes were mixed with apomyoglobin at 5x the concentration of MhuD. Spectrally, this gave rise to a species with a greater Soret intensity and altered spectral features compared with the heme-MhuD complexes (Figure 6). In contrast to data presented by Chao and Goulding 31, where transfer of heme

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from MhuD to apo-H64Y/V68F-myoglobin was measured over 30 minutes to give a dissociation rate of 0.0015 min–1, we found that two separate heme dissociation events occur upon addition of apomyoglobin to heme-bound MhuD (Figure 6).

The first heme dissociation event is mostly complete within 30 seconds, and the rate constant was determined by stopped-flow kinetics. Stopped-flow measurements show spectral data with clear isosbestic points at 420, 493, 515 and 604 nm. The dissociation and transfer of heme from MhuD to apomyoglobin was plotted at a wavelength of 407.5 nm, and data were fitted using a single exponential function. Average heme dissociation rates were similar across the MhuD-heme stoichiometries tested, with values of 0.22, 0.25 and 0.25 s-1 for 5 µM heme in complex with 1.25, 2.5 and 5 µM dimeric-MhuD, respectively. The second heme dissociation rate was much slower, and heme transfer was measured over one hour using UV-visible spectroscopy. These spectra have isosbestic points mostly distinct from those for the first heme dissociation event, at wavelengths of 379, 430, 495, 546 and 634 nm. Heme dissociation and transfer kinetics were plotted at 410 nm to give dissociation rate constants of 0.125, 0.121 and 0.103 min-1 for 5 µM heme in complex with 1.25, 2.5 and 5 µM dimeric-MhuD, respectively.

Although the rates of heme transfer were similar across the different MhuD-heme stoichiometries tested, the amplitudes of the two heme dissociation events varied significantly (Table 2). When the heme:dimeric-MhuD stoichiometry was 1:1 the most populated heme-MhuD species is the monoheme form (according to earlier MS studies), the fast heme dissociation rate has a >2-fold amplitude compared with the heme:MhuD stoichiometry of 4:1 (where MhuD is mainly in a 4-heme bound state). Conversely, the amplitude of the slow heme transfer event was decreased by a factor of >2 for the 1:1 complex compared with the 4:1 heme:MhuD stoichiometry. Analysis of the 2:1 heme-MhuD complex, which is made up of apo-, 1-, 2- and 3-heme bound forms of MhuD, showed that the amplitude of the fast phase was roughly halfway between, and the amplitude of the slow phase was comfortably between, those of the 1:1 and 4:1 stoichiometries. On the basis of these data, we propose that dissociation of diheme from MhuD is a much slower event compared with that of the dissociation of monoheme.

It is well known that MhuD can degrade heme 29,38, and it is clear from structural data that MhuD can bind up to four heme molecules and act as a heme storage protein 24. Previous reports have indicated that binding of monoheme and diheme are regulated through tight binding (nM Kd values) of the first heme molecule to the His75 residue, and that higher heme concentrations (µM Kd values) allow binding of a second heme molecule in order to sequester heme and prevent toxicity associated with free heme (Figure 7A) 39. These studies employed fluorescence spectroscopy with the aim of measuring Förster resonance energy transfer (FRET) quenching of the active site Trp66 residue by addition of the first heme substrate, and absorbance spectroscopy to measure binding of the second heme. Data presented here contradict this model, as the MS binding data show polydispersity of heme-MhuD stoichiometries at concentrations where the previous model would predict the presence of only monoheme-MhuD. Additionally, aIM-MS studies showed that diheme binding did occur in the 2-heme-bound MhuD, indicating that one of the MhuD monomers would have been in the apo-form - a phenomenon that could not be explained if the Kd of monoheme was 103 times tighter than that of the second heme molecule.

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Our MS results describe a system where the binding affinities of the solvent accessible and solvent protected hemes are less distinct. The modelled Kd values derived from our data show that formation of the monoheme-MhuD species is preferred, but only modestly compared with binding of a second heme to form the diheme complex. The aIM-MS data also indicated that MhuD-bound diheme is tightly complexed with a molecule of water, and that the interactions between the two heme molecules are stronger than the interactions between the heme molecules and the MhuD active site. Through apomyoglobin heme transfer assays, we have also shown that the diheme complex dissociates at a slower rate from MhuD than does the monoheme form. In combination, we believe these data present a heme binding mechanism where the Kd of the second heme is only slightly weaker than that of the first heme molecule (see “Modelling of Kd values for MhuD-heme binding” section below). In this model, binding of the second heme within the MhuD active site would bring the two heme molecules together with a molecule of water; enhancing the π-π interactions between the two hemes. This diheme complex would become tightly sequestered within the MhuD active site (Figure 7B), decreasing the levels of free heme within the Mtb cell.

The importance for Mtb to maintain tightly regulated iron levels is clear (Figure 8). Import of ferric iron from the host to the Mtb cell is mediated by the siderophores carboxymycobactin and mycobactin 40–43. These siderophores function with the assistance of MmpS4/MmpL4 and MmpS5/MmpL5 which are involved in carboxymycobactin export 44, and the IrtAB transporter which mediates iron internalization 40,45. Excess iron is toxic, and is therefore sequestered by ferritins BfrA (utilizes heme) and BfrB (does not bind heme), which aggregate to form macromolecular structures that exhibit ferroxidase activity, and contain a hollow internal cavity in which the iron mineral is stored (45-48).

Heme is also vital for Mtb, and is required for the function of essential hemoproteins (Figure 8). Mtb is able to synthesize heme in a process involving the ferrochelatase HemZ 50, as well as importing heme through a pathway involving the Mtb PPE37, Rv0203, PPE36, PPE62 and Rv0265c proteins (50-52). Currently, MhuD is the only mycobacterial protein known to sequester and store heme. The ability of MhuD to also degrade heme allows it to not only regulate free heme levels in the cell, but also to bridge both heme and iron regulatory networks. Upon high cellular heme levels, MhuD sequesters heme as diheme, which can be gradually released under decreased cellular heme conditions. When heme levels in the cell are adequate, heme is degraded by MhuD to release free iron, contributing to iron homeostasis. However, when heme levels fall too low, MhuD will not bind heme. This ensures that heme does not continue to be degraded at low concentrations, and therefore the heme pool remains sufficient to supply heme-requiring proteins. We hypothesize that data reported here are consistent with a heme storage and release role for MhuD that provides adequate levels of cellular heme to the Mtb pathogen, in addition to a heme degradation function that provides iron to the Mtb cell.

ConclusionsMhuD is unique among heme oxygenases in that it is able to bind four heme molecules simultaneously. It has been proposed that this provides an evolutionarily economical strategy of providing heme storage under non-iron depleted conditions 24. In this manuscript we present data exploring the stoichiometry of heme binding to MhuD, and have found that the strength of binding

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of a second heme molecule is only modestly different to that of the first heme molecule. This indicates that MhuD has not evolved to preferentially bind one heme molecule in each monomer. In diheme-MhuD, the two heme molecules are tightly stacked, with the heme irons coordinated by water and imidazole nitrogen. This complex is bound tightly within the MhuD active site and displays tighter binding compared with a single heme molecule. These data suggest that MhuD plays an important role in tightly regulating heme levels to ensure adequate supply of heme to essential Mtb hemoproteins, whilst reducing the toxicity mediated by excess heme and contributing to the free iron pool.

Methods

Expression and Purification of MhuDThe gene encoding MhuD from M. tuberculosis (strain ATCC 25618 / H37Rv) was codon optimized for expression in E. coli (Thermo Fisher Scientific GENEART GmbH, Germany), and cloned into pET28b to provide a construct with an N-terminal polyhistidine tag followed by a TEV cleavage site.

This plasmid was transformed into E. coli strain BL21 gold (DE3), and cells were grown at 37°C with shaking at 190 RPM in 500 mL cultures of LB containing 30 µg/mL kanamycin. At an OD600 of 0.6–0.8, mhuD gene expression was induced with 100 µM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the temperature was lowered to 25°C. After 16 hours, cells were harvested by centrifugation at 6000 rpm at 4°C using a JLA-8.100 rotor in an Avanti J-26 XP centrifuge. The pellet was frozen at -20°C

When required, the cell pellet was defrosted and resuspended in 200 g/L extraction buffer (100 mM sodium phosphate, 250 mM NaCl, 20 mM imidazole, 2 mM magnesium chloride, pH 8.0) containing a SigmaFAST protease inhibitor tablet (EDTA-free, 1 tablet/100 mL), 10 µg/mL lysozyme (hen egg white, Merck, Poole, UK) and 20 µg/mL DNase I (bovine pancreas, Merck).

Cells were lysed on ice, using a Bandelin Sonopuls sonicator. At 20% amplitude, the suspension was exposed to 10 s pulses, at 60 s intervals, for a total of 10 minutes. The homogenate was then centrifuged at 20,000 rpm at 4°C for 1 hour using a JA-25.50 rotor (Beckman-Coulter Ltd., High Wycombe, UK). The supernatant was filtered through a 0.2 µm filter membrane (Sartorius, Göttingen, Germany), before loading onto a 5 mL HisTrap FF column (GE Healthcare, Little Chalfont, UK). The column was then washed with 20 column volumes of buffer A (100 mM NaPi, 250 mM NaCl, pH 8) containing 20 mM imidazole, followed by 5 column volumes of buffer A containing 40 mM imidazole and then 5 column volumes of buffer A containing 60 mM imidazole. MhuD was then eluted with buffer A containing 250 mM imidazole.

The eluted protein was exchanged into buffer A using a HiPrep 26/60 desalting column (GE Healthcare). The S219V variant of TEV protease was expressed using plasmid pRK793 (a gift from David Waugh, addgene plasmid no. 8827) 54, and MhuD was incubated overnight with TEV protease in a ratio of 1 mg TEV to 5 mg MhuD. The following day, reverse nickel column purification was performed to separate non-cleaved MhuD. The overnight incubation was loaded onto a 5 mL HisTrap FF column pre-equilibrated with buffer A, and cleaved MhuD was eluted with buffer A containing 30 mM imidazole.

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TEV-cleaved MhuD was then concentrated using a Vivaspin with a 5 kDa MWCO (GE Healthcare), and loaded onto a HiLoad 26/600 Superdex 75 pg column (GE Healthcare) that had been pre-equilibrated with buffer B (100 mM KPi, 250 mM NaCl, pH 7.5). Fractions containing pure MhuD were then pooled and aliquoted, before being snap-frozen in liquid nitrogen, and then stored at -80°C until use.

Protein destined for native mass spectrometry was purified as described above. However, the protein sample was not gel filtered into buffer B. Instead, the protein was loaded onto a HiLoad 26/600 Superdex 75 pg column (GE Healthcare) that was pre-equilibrated with buffer C (100 mM ammonium acetate, pH 7). Fractions containing pure MhuD were then pooled and loaded directly onto the same column. MhuD was then pooled and diluted to a concentration of 5 µM dimeric MhuD. Porcine hemin (Merck) was dissolved in DMSO and added to MhuD samples to give a final DMSO concentration of 0.5%. Samples were incubated overnight at 4°C, before analysis by native mass spectrometry the following day. Control native mass spectrometry experiments were conducted after a 1 hour incubation of 5 µM dimeric MhuD with 10 µM heme (0.5% DMSO) at room temperature, and then repeated following overnight incubation at 4°C. The resulting spectra were almost identical, indicating that overnight incubation of MhuD with heme had no effect on heme binding stoichiometry (Figure S7).

Native Mass Spectrometry (MS) and Ion Mobility MS (IM-MS)Native MS data were acquired on a Synapt G2S HDMS instrument (Waters, Manchester, UK). NanoESI capillaries were prepared in-house from thin-walled borosilicate capillaries (inner diameter 0.9 mm, outer diameter 1.2 mm, World Precision Instruments, Stevenage, UK) using a Flaming/Brown P-1000 micropipette puller (Sutter Instrument Company, Novato, CA, USA). A positive voltage was applied to the solution via a platinum wire (Goodfellow Cambridge Ltd, Huntington, UK) inserted into the capillary. Gentle source conditions were applied to preserve the native-like structure: capillary voltage 1.2-1.5 kV, sampling cone 50-100 V, source temperature 70°C. Trap collision energy was 4 V, transfer collision energy was set to 0 V. Nitrogen was the carrier gas. For the IM, the helium cell and the IMS gas flows were 180 and 90 mL/min, respectively; the IMS wave velocity was 400 m/s, and the IMS wave height was 35 V. External mass calibration of the spectra was achieved using solutions of sodium iodide (2 mg/mL in 50:50 water:isopropanol). TWCCSN2 calibration was carried out using β-lactoglobulin. Data were acquired and processed with MassLynx software (Waters). Mass spectra were deconvoluted using UniDec software 55.

Activated Ion Mobility Mass Spectrometry (aIM-MS) Experiments were performed on a Waters Synapt G2S instrument using nESI and trap-activated ion mobility; capillary voltage 1.2 – 1.5 kV, cone 50 V and source temperature 70oC. The helium cell and the IMS gas flows were 180 and 90 mL/min, respectively; the IMS wave velocity was 400 m/s, and the IMS wave height was 35 V. Nitrogen was the carrier gas. The most intense charge state [M+8H]8+

for each protein species was mass selected using the quadrupole prior to the trap region. Activation was induced by elevating the trap collision energy. ORIGAMI 33 was used to automatically acquire data for collision energies from 4 – 200 V in 2 V increments, as well as for data processing.

Modelling of Kd values for MhuD-heme bindingData were modelled numerically using a sequential binding model with the four Kd values and the total enzyme concentration defined:

10

E+H⇌Kd ,1

E . H+H⇌Kd , 2

E . H 2

+H⇌Kd ,3

E .H 3

+H⇌Kd ,4

E . H 4

[H ]=[ totalheme ]−([E . H ]+2 [E . H2 ]+3 [E . H 3 ]+4 [E .H 4 ] )

[ totalenzyme ]=[E ]+[E .H ]+ [E . H2 ]+ [E .H3 ]+[E . H 4 ] (1)

In all cases, the total (dimeric) enzyme concentration was fixed to the experimental value of 5 μM. Modelling was performed using the NSolve function in Mathematica version 11.2 (Wolfram Research Inc.). The model could not be practically used to fit the data, but can be used to estimate the four Kd values. The apparent Kd value of 1.3 ± 0.7 µM determined from the Morrison equation fit of the fraction of heme-bound protein was use to restrain the individual Kd’s, such that:

K d ,app=Kd ,1 .K d ,2 .K d ,3 . Kd , 4 1µM

In the simplest case, the four Kd values were set to be equal (1 μM). However, this does not adequately describe the data, with the five modelled species all crossing (having equal concentration) at 11 μM; behaviour that is not observed experimentally. Instead, the four individual Kd values were estimated from the ESI-MS data collected at 10 µM hemin concentration. Under these conditions, apo-MhuD plus all four heme-bound MhuD species are observed. This allows the free hemin (H in Eq 1) to be determined, which in turn allows determination of all 4 Kd values by e.g. Kd1 = [E][H]/[E.H]. The data in Figure 3A were then modelled using Eq 1 with these 4 Kd values, which are 0.45, 0.55, 1.1 and 2.2 µM, respectively.

Analytical UltracentrifugationApo-MhuD was gel filtered into 100 mM NaPi, 250 mM NaCl (pH 7.5) buffer using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare), and diluted to concentrations of 1 and 5 μM before being loaded into 2-sector Epon filled centerpieces with quartz glass windows. Samples were monitored at 230 nm and sedimentation was achieved by running at 50,000 rpm collecting 300 scans every 1.5 minutes. Data were analysed using Sedfit56, and output using GUSSI57.

Apomyoglobin assaysApomyoglobin assays were performed with apomyoglobin purchased from Merck. Various concentrations of MhuD (2.5, 5, 10 µM dimeric MhuD) were incubated with hemin (at a final concentration of 10 µM) overnight at 4°C in buffer D (20 mM KPi, pH 7).

For full analysis of heme transfer, data were collected at 407.5 nm using a Cary 60 UV-visible spectrophotometer. Assays were initiated through mixing equal volumes of heme-bound MhuD with apomyoglobin (suspended in buffer D) prepared at a concentration 10 times that of dimeric MhuD. Consequently, post-mixing concentrations were as follows: 1.25 µM MhuD + 5 µM hemin vs 12.5 µM apomyoglobin; 2.5 µM MhuD + 5 µM hemin vs 25 µM apomyoglobin; and 5 µM MhuD + 5 µM hemin vs 50 µM apomyoglobin. Measurements were taken in triplicate. Transfer of heme to apomyoglobin was monitored over 1 hour.

11

For shorter time scales, the same experiment was performed with data collected over 30 seconds using an Applied Photophysics SX18 MR stopped-flow spectrophotometer with a photodiode array detector (Leatherhead, UK).

AbbreviationsaIM-MS, activated ion mobility mass spectrometry; AUC, analytical ultracentrifugation; IM-MS, ion mobility mass spectrometry; MhuD, mycobacterial heme utilization, degrader; Mtb, Mycobacterium tuberculosis; nESI-MS, nanoelectrospray mass spectrometry

Conflict of InterestThe authors declare no competing financial interest.

Acknowledgements The authors acknowledge financial support through funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC) through grant award BB/P010180/1 to AWM and SJM. The authors are also grateful for technical support from Mrs Marina Golovanova (University of Manchester) and for useful discussions and guidance from Dr Hazel Girvan, Dr Kirsty McLean and Dr Harshwardhan Poddar (University of Manchester). The authors also acknowledge the BBSRC-funded Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM, BB/M017702/1) for access to analytical equipment.

Supporting InformationFigure S1. SDS-PAGE gel from a MhuD purificationFigure S2. Analysis of MhuD dimeric state using analytical ultracentrifugation (AUC)Figure S3. Absolute and deconvoluted nESI-MS spectra over various hemin concentrationsFigure S4. Collision cross section distributions (TWCCSDN2) for apo- and heme-bound MhuD [M + 8H]8+

charge statesFigure S5. Absolute IM-MS spectrum of 10 M MhuD + 10 M deuteroheme (+0.5% DMSO)Figure S6. Absolute IM-MS spectrum showing heme species formed through aIM-MS analysis of 2-heme-bound MhuD (m/z 2982)Figure S7. Effect of heme-MhuD incubation times on nESI-MS spectra

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Figure 1. Overlaid structure of monoheme-CN-MhuD and diheme-MhuDThe figure shows overlaid structures of monoheme-CN-MhuD depicted in yellow (PDB code 4NL5) and diheme-MhuD in pink (PDB code 3HX9). Panel A shows the full dimeric structures of MhuD, where dimeric diheme-MhuD is able to bind up to four molecules of heme in the inactive state. A dashed box highlights the active site of one MhuD monomer, which is shown in more detail in Panel B. Panel B shows that, in monoheme-CN-MhuD, heme iron is ligated through His75. In the diheme-MhuD structure, the solvent exposed heme is ligated to His75, with the solvent protected heme stacked to occupy roughly the same position as the single heme molecule in monoheme-CN-MhuD. By overlaying the two structures, we can see that MhuD is able to accommodate two heme molecules in one active site by extending the kink at Asn68 in the α2-helix.

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Figure 2. Absolute and deconvoluted nESI-MS spectra depicting heme-MhuD binding stoichiometries. nESI-MS spectra of 5 µM MhuD in 100 mM ammonium acetate + 0.5% DMSO (A, B + C) and 5 µM MhuD + 10 µM hemin in 100 mM ammonium acetate buffer + 0.5% DMSO (D + E) samples at pH 7.3. The spectra in panels B and C show the [M + 9H]9+, [M + 8H]8+ and [M + 7H]7+ MhuD charge states. The heme-free sample shown in panel A only has one species of each charge state relating to apo-MhuD (purple circles). In addition to the apo-protein, the heme-containing sample in panel D also contains 1-, 2-, 3- and 4-heme bound species (blue triangles, cyan triangles, orange triangles and red squares). Panels C and E show the deconvoluted spectra including peak areas used to calculate the abundance of the different heme-MhuD stoichiometries.

18

[M+9H

]9+

[M+8H]8+

[M+7H]7+

[M+9H]9+

[M+7H]7+

A

Apo MhuDC

MhuD+ 4 heme

MhuD+ 3 heme

MhuD+ 2 heme

MhuD+ 1 heme

MhuDapo

E

[M+8H]8+B

Dimeric MhuD

D

Figure 3. Determination of heme binding to MhuD using ESI-MS. Panel A shows the relative amounts of apo- (purple circles), 1- (blue triangles), 2- (cyan triangles), 3- (orange triangles) and 4- (red squares) heme-bound forms of MhuD at different hemin concentrations, as determined from the deconvoluted peak areas obtained by ESI-MS. Data were modelled numerically (solid lines) using a sequential binding model with the four Kd values given in the main text. Panel B shows the relative total heme bound to MhuD (normalised sum of all four heme-bound species) at different heme concentrations. These data were fitted using the Morrison equation with an adjusted R2 value of 0.996, providing an apparent Kd of 1.3 ± 0.7 µM and an apparent binding site heme concentration of 18.4 ± 1.5 M.

19

Figure 4. 2D activated ion mobility MS plots of apo- and heme-bound MhuD. Collision-induced activation of apo-, 1-, 2-, 3- and 4-heme bound MhuD (panels A, B, C and D, respectively) gives rise to two main unfolding events depicted by dashed lines. Additionally, apo- and 1-heme bound MhuD (panels A and B) dissociate to form monomeric MhuD (shown in dotted boxes) as the collision voltage increases.

20

Figure 5. RT and 1D plots showing MhuD and heme species from aIM-MS analysis of 2-heme bound MhuD (m/z 2982). Panel A shows how increasing collision voltage affects the population of MhuD and its heme bound forms throughout the aIM-MS experiment. Elevating voltage resulted in removal of the water molecule from diheme and subsequent dissociation of diheme to form monomeric heme. Panel B demonstrates the drift times of heme, diheme, diheme + water and MhuD, which, in particular, highlights the more compact structure of diheme when complexed with a water molecule, as compared with the water-free diheme species.

21

Figure 6. Apomyoglobin competition assays to determine koff for heme-MhuD binding. Panels A and B show spectra taken at T0 (black line) and T20s (dashed blue line) with derived difference spectra (gray line) after mixing 5 µM MhuD + 5 µM heme with 50 µM apomyoglobin, and 10 µM MhuD + 5 µM heme with 100 µM apomyoglobin, respectively. Panels C and D show spectra taken at T1min (dashed blue line) and T30min (red line) for samples detailed in panels A and B, respectively (again with derived difference spectra). The insets show data at 407.5 nm (A and B) and 410 nm (C and D) and are plotted with data fitted using a single exponential function with Origin software.

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A

DC

B

Figure 7. Proposed models illustrating how MhuD binds heme to regulate heme storage and heme oxygenase activity.Panel A describes the model proposed by Thakuri et al (38), where a single molecule of heme (cyan) is proximally coordinated by an imidazole nitrogen atom and binds MhuD with a nanomolar Kd, which allows MhuD to degrade heme at low concentrations. At higher heme concentrations, MhuD will bind a second molecule of heme (gold) with a micromolar Kd in order to decrease the cellular heme concentration. Panel B shows the model supported by data reported in our study, where the binding of a second heme molecule forms a diheme complex (pink) with the second heme coordinated to a distal water molecule that bridges to a nitrogen atom on Asn7. The diheme complex dissociates more slowly from MhuD than does the monoheme species. A chloride ion was found to coordinate the solvent-protected heme in the MhuD diheme crystal structure (24), but our aIM-MS analysis indicates that a water molecule is bound instead in the solution state. The PDB structures shown are for cyanide-coordinated monoheme (PDB 4NL5) and diheme (PDB 3HX9) forms of MhuD.

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A

B

Figure 8. A model describing how MhuD contributes to heme and iron homeostasis in M. tuberculosis.As described in the text, MhuD is able to regulate heme levels in Mtb to ensure adequate supply for hemoproteins, in addition to preventing heme-mediated toxicity. Heme can be synthesised by Mtb in a pathway involving the ferrochelatase HemZ, which catalyzes the incorporation of Fe2+ into protoporphyrin IX to give protoheme 50. Heme can also be imported from the host and into the cell via the cell membrane associated PPE36, PPE62, PPE37, periplasmic associated Rv0265c, and extracellularly secreted heme scavenging protein Rv0203 51–53. MhuD can contribute to the pool of cellular iron through catalyzing heme degradation. Levels of free iron can also be regulated though iron import from the host in a siderophore-mediated pathway. In this pathway, MmpS4/MmpL4 and MmpS5/MmpL5 export the iron scavenging siderophore carboxymycobactin, which subsequently binds host iron and transfers the iron to the cell envelope restricted mycobactins 40–44. Iron internalization is also mediated by the membrane associated IrtAB transporter 40,45. Iron toxicity is prevented by iron storage ferritins BfrA and BfrB, which sequester excess iron 46–49. The figure highlights, with blue arrows, the role of MhuD in maintaining heme/iron homeostasis.

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Charge state CCS / nm2 Average CCS / nm2

apo 9+ 15.55 15.48 ± 0.218+ 15.25

7+ 15.64

+ 1 heme 9+ 16.08 15.79 ± 0.308+ 15.47

7+ 15.82

+ 2 heme 9+ 16.34 16.14 ± 0.218+ 15.91

7+ 16.18

+ 3 heme 9+ 16.59 16.49 ± 0.138+ 16.35

7+ 16.52

+ 4 heme 9+ 16.85 16.77 ± 0.188+ 16.56

7+ 16.88

Table 1. Median collisional cross sections (TWCCSN2) for apo-, 1-, 2-, 3- and 4-heme bound MhuD. The table shows the collision cross sections (TWCCSN2) of [M + 9H]9+, [M + 8H]8+ and [M + 7H]7+ charge states for apo, 1-, 2-, 3- and 4-heme bound MhuD, as well as the average TWCCSN2 across the charge states.

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5 µM Heme5 µM MhuD (1 heme: 1 MhuD dimer)

5 µM Heme2.5 µM MhuD (2 heme: 1 MhuD dimer)

5 µM Heme1.25 µM MhuD (4 heme: 1 MhuD dimer)

k1 0.253 ± 0.003 s-1 0.246 ± 0.002 s-1 0.222 ± 0.003 s-1

A1 0.104 ± 0.001 0.073 ± 0.001 0.047 ± 0.001

k2 0.103 ± 0.005 min-1 0.121 ± 0.006 min-1 0.125 ± 0.016 min-1

A2 0.096 ± 0.007 0.221 ± 0.011 0.247 ± 0.014

Table 2. Rates and amplitudes of the two heme dissociation and transfer events from MhuD to apomyoglobin. A1 (amplitude) and k1 (rate constant) values from the fast heme dissociation phase were determined using stopped-flow kinetics; and A2 and k2 values from the slow heme transfer phase were obtained using steady-state UV-visible spectroscopy. Data were fitted using a single exponential function, and experiments were performed in triplicate to obtain standard errors.

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