Protein/Protein Interactions in the Mammalian Heme Degradation Pathway: Heme Oxygenase-2, Cytochrome P450 Reductase, and Biliverdin Reductase

Heme oxygenase (HO) catalyzes the rate-limiting step in the O2- dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of bili­verdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, im­plicating these residues at the HO/CPR interface. Alanine substitu­tions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescenc

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway: Heme Oxygenase-2, Cytochrome P450 Reductase, and Biliverdin Reductase

Heme oxygenase (HO) catalyzes the rate-limiting step in the O2- dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of bili­verdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, im­plicating these residues at the HO/CPR interface. Alanine substitu­tions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescenc

Not Just a Passive Adaptor, the Periplasmic Component CusB of Escherichia coli's CusCFBA Copper Efflux System has an Active Functional Role

Increased emergence of antibiotic resistance in bacterial pathogens has posed a serious threat to human health. Due to similar structural and functional characteristics of metal and antibiotic resistance systems in gram-negative bacteria, there is a growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. The CusCFBA copper/silver resistance system of Escherichia coli forms a protein complex that spans the inner and outer membranes and functions in the efflux of metal from the periplasm to the extracellular space. In order to understand the molecular details of metal resistance by the Cus system and more specifically, to define the role of the periplasmic components in CBA type metal transporters, I characterized CusB and probed its interactions with CusF using various structural and biochemical tools. CusB was previously thought to play a relatively passive role as an adaptor protein that stabilized the association of the inner and outer membrane proteins. Through isothermal titration calorimetry (ITC), X-ray absorption spectroscopy (XAS), and mutagenesis, I have shown that CusB binds Cu(I)/Ag(I) with high affinity using three conserved methionines. Gel filtration chromatography experiments showed that upon binding Ag(I), CusB undergoes a substantial conformational change. Importantly, functional metal binding by CusB is essential for cell survival in environments with elevated copper concentrations. The small periplasmic metal binding protein CusF is a unique component of monovalent metal resistance systems serving an unknown function. To determine the nature and specificity of interaction between CusF and CusB, ITC and NMR were used to show that the interaction between CusF and CusB is metal-dependent and specific for the components of Cus system. From NMR chemical shift perturbations, the CusB interaction face on CusF was determined to overlap with the metal binding site. XAS experiments demonstrate metal transfer between CusB and CusF, which supports the role of CusF as a metallochaperone. In summary, these findings demonstrate an active role for CusB in metal resistance, and suggest that the plausible role for CusF is that of a metallochaperone for CusB

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway \u3ci\u3eHEME OXYGENASE-2, CYTOCHROME P450 REDUCTASE, AND BILIVERDIN REDUCTASE\u3c/i\u3e

Background: Heme oxygenase, cytochrome P450 reductase, and biliverdin reductase are the key enzymes in heme degradation. Results: Specific electrostatic and hydrophobic interactions form the binding interface between heme oxygenase and cytochrome P450 reductase. Conclusion: Heme oxygenase binds cytochrome P450 reductase dynamically and biliverdin reductase very weakly. Significance: Characterizing interactions among proteins involved in heme degradation are crucial to understanding heme homeostasis

The C‑Terminal Heme Regulatory Motifs of Heme Oxygenase‑2 Are Redox-Regulated Heme Binding Sites

Heme oxygenase-2 (HO2), an enzyme that catalyzes the conversion of heme to biliverdin, contains three heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1–288 (HO2_<i>sol</i>) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2_<i>sol</i> via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe³⁺-heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of heme to disulfide-reduced HO2_<i>sol</i>, in the work described here, we further investigated the properties of Fe³⁺-heme bound to HO2. Specifically, we investigated binding of Fe³⁺-heme to a truncated form of soluble HO2 (residues 213–288; HO2_<i>tail</i>) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2_<i>tail</i> in the disulfide-reduced state binds Fe³⁺-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2_<i>sol</i> that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2_<i>sol</i> binds one Fe³⁺-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2_<i>sol</i> binds slightly more than two. Both Cys265 and Cys282 were identified as Fe³⁺-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe³⁺-heme binds with a much weaker affinity to Cys282 than to Cys265, which has an affinity much weaker than that of the His45 binding site in the catalytic core. In summary, disulfide-reduced HO2 has multiple binding sites with varying affinities for Fe³⁺-heme

Spectroscopic Studies Reveal That the Heme Regulatory Motifs of Heme Oxygenase‑2 Are Dynamically Disordered and Exhibit Redox-Dependent Interaction with Heme

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2^O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2^R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme