Heme binding to the SARS-CoV-2 spike glycoprotein.

The target for humoral immunity, SARS-CoV-2 spike glycoprotein, has become the focus of vaccine research and development. Previous work demonstrated that the N-terminal domain (NTD) of SARS-CoV-2 spike binds biliverdin-a product of heme catabolism-causing a strong allosteric effect on the activity of a subset of neutralizing antibodies. Herein, we show that the spike glycoprotein is also able to bind heme (KD = 0.5 ± 0.2 µM). Molecular modeling indicated that the heme group fits well within the same pocket on the SARS-CoV-2 spike NTD. Lined by aromatic and hydrophobic residues (W104, V126, I129, F192, F194, I203, and L226), the pocket provides a suitable environment to stabilize the hydrophobic heme. Mutagenesis of N121 has a substantive effect on heme binding (KD = 3000 ± 220 µM), confirming the pocket as a major heme binding location of the viral glycoprotein. Coupled oxidation experiments in the presence of ascorbate indicated that the SARS-CoV-2 glycoprotein can catalyze the slow conversion of heme to biliverdin. The heme trapping and oxidation activities of the spike may allow the virus to reduce levels of free heme during infection to facilitate evasion of the adaptive and innate immunity.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV=O or FeIV-OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel.

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

Solution structure of the cytochrome P450 reductase-cytochrome c complex determined by neutron scattering.

Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein-protein complexes.

Orchestrated Domain Movement in Catalysis by Cytochrome P450 Reductase.

NADPH-cytochrome P450 reductase is a multi-domain redox enzyme which is a key component of the P450 mono-oxygenase drug-metabolizing system. We report studies of the conformational equilibrium of this enzyme using small-angle neutron scattering, under conditions where we are able to control the redox state of the enzyme precisely. Different redox states have a profound effect on domain orientation in the enzyme and we analyse the data in terms of a two-state equilibrium between compact and extended conformations. The effects of ionic strength show that the presence of a greater proportion of the extended form leads to an enhanced ability to transfer electrons to cytochrome c. Domain motion is intrinsically linked to the functionality of the enzyme, and we can define the position of the conformational equilibrium for individual steps in the catalytic cycle.