Characterization of Heme Ligation Properties of Rv0203, a Secreted Heme Binding Protein Involved in Mycobacterium tuberculosis Heme Uptake

Abstract

The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work we use spectroscopic (absorption, electron paramagnetic resonance and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low spin in both ferric and ferrous oxidation states. Rv0203-notag is high spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen ligated heme proteins. Mutagenesis experiments identified that residues Tyr59, His63 and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63 and His89 are required for heme binding to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggest the heme-ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for heme binding to Rv0203-His and Rv0203-notag are similar, 115 (μM s)-1 and 133 (μM s)-1, respectively. However, the heme off-rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 s-1 and 0.0002 s-1, respectively, and Rv0203-notag has a single off-rate of 0.082 s-1. The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His-tag interferes with heme binding. Furthermore, these results imply that the His-tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins implying that Rv0203 may act as an extracellular heme transporter.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

“…Biochemical studies indicate that heme dissociates from Rev-erb␤ too slowly to equilibrate with the heme pool and thus would be unable to act, as suggested previously (16,49), as an intracellular heme sensor. We also demonstrate that heme has only a minimal effect on the binding of NCoR1 to FLRev-erb␤, refuting our original hypothesis that full-length proteins could reproduce the effects of heme observed in the cell.…”

“…In some cases, the affinity is so high that even the quadratic treatment does not provide correct K d values. For example, a recent report from our laboratory showed that the K d for Fe 3ϩ -heme binding to heme oxygenase-2 determined with stoichiometric equilibrium titrations was 70-fold higher (14 nM) than the value (0.2 nM) calculated from heme on-and off-rates (45 For the reasons just discussed, we measured the Fe 3ϩ -heme off-rates, the controlling factor in distinguishing heme affinity among a variety of heme proteins (49,75). We mixed FLReverb␤⅐Fe 3ϩ -heme complexes with 10-fold excess apomyoglobin and monitored the conversion of apo-to holomyoglobin at 408 nm ( Fig.…”

Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease

“…The final purification step was performed by using an S200 10/30 Superdex column (GE Healthcare) equilibrated with a solution containing 50 mM Tris (pH 7.4) and 150 mM NaCl to yield 100% homogeneous Hal N . The percentage of heme was determined by using the pyridine hemochrome and Lowry assays (Bio-Rad) (64,65). To achieve fully heme-reconstituted Hal N , a hemin solution was added to a 1:1 molar ratio of heme to protein (Sigma) (64,65).…”

“…The percentage of heme was determined by using the pyridine hemochrome and Lowry assays (Bio-Rad) (64,65). To achieve fully heme-reconstituted Hal N , a hemin solution was added to a 1:1 molar ratio of heme to protein (Sigma) (64,65). Crystals of heme-Hal N were grown at 90 mg/ml by a hanging-drop vapor diffusion method with a reservoir containing 0.2 M ammonium sulfate, 0.1 M BisTris (pH 5.5), and 21% polyethylene glycol 3350 (PEG 3350) with a 1:1 (vol/vol) protein-to-reservoir drop.…”

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

“…Solutions were made, and titration experiments were performed as described previously (21,22). Heme (23) and PPIX (24) solutions were freshly prepared prior to each experiment.…”