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

The ether-à-go-go (EAG) 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 amino-terminus, known as the eag-domain, consisting of a 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 pull-down 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 E.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.


INTRODUCTION
PAS (Per-ARNT-Sim) domains, first identified by sequence homology in the Drosophila proteins period and single-minded, and the vertebrate aryl hydrocarbon receptor nuclear transporter (ARNT), are well known sensory modules present in a variety of signalling proteins in organisms ranging from bacteria to humans (1)(2)(3). PAS domains demonstrate considerable plasticity in binding different biologically-relevant ligands. Relevant in the context of this work is their ability to bind heme as a ligand. The ligand binding event is significant as it can act as a primary trigger to initiate a cellular signalling / regulatory response; or, it can provide the PAS domain with a capacity to respond to secondary physical or chemical signals (such as binding of gas molecules, changes in redox potential, or light activation). In the case of heme as a ligand, there are several examples of heme-binding PAS domains that act as biological regulators in cells, for example the O2 sensor proteins (e.g. FixL, E. coli DOS) and the transcriptional regulators involved in circadian control (e.g. nPAS2, CLOCK, Rev-ERB, Per) (4,5).
Several layers of regulatory control can be envisaged in the process of heme binding to a PAS domain protein. Heme binding might, for example, induce a conformational change in the PAS domain which affects the interactions with partner proteins, and there is evidence for this in the heme-binding circadian proteins (6). And since known cell sigalling gases such as carbon monoxide (CO), nitric oxide (NO) and dioxygen (O2) all bind to heme (and with different affinities), then the formation of a heme-bound PAS domain opens opportunities for the molecule to also respond to changes in concentrations of any or all of these cell signalling gases. Thinking in these terms, it may not be a coincidence that CO and NO production in cells is catalysed by two heme-containing proteins -heme oxygenase and nitric oxide synthase, respectively -both of which are themselves O2-dependent. This interplay of heme-and gas-responsive PAS domains could provide the cell with highly versatile mechanisms for regulatory control, that are at the same time responsive to changing concentrations of heme, O2, CO or NO. How these mechanisms play out has yet to be fully established. In this paper, we present evidence from electrophysiological experiments for hemedependent modulation of hERG3 (human ether-àgo-go related gene 3, alternatively known as Kv11.3). We identify an interaction of heme with a PAS domain contained within the channel. Our results provide insight on the role of heme in channel regulation; we use this to discuss how heme-induced structural changes might be used to control channel function .

Proteomics analyses.
We sought to identify potential heme binding proteins in neuronal lysates from primary cortical neurons, using heme-affinity chromatography and mass spectrometry. Using this approach, we identified a number of proteins known to bind heme (e.g. mitochondrial enzymes) as well as proteins with unknown heme affinity; this included hERG3 (Fig. S1). In the structure of the overall hERG3 channel, there are four subunits each containing a transmembrane region as well as cytoplasmic Nand C-terminal regions, Fig. 1 (7). The N-terminal cytoplasmic region (approximately 400 residues) contains a highly conserved eag-domain (residues 1-135) which itself comprises a Cap domain (residues 1-25) and a Per-ARNT-Sim (PAS) domain (residues . Heme binding to the hERG3-eag domain. We expressed the Cap domain (residues 1-26) and the PAS  domain of the hERG3 protein as an N-terminal His6/S-tagged fusion protein in E.coli (Figs. 1 and S2); we will refer to this protein as hERG3-eag. On addition of heme to hERG3-eag, Fig. 2A, a species with Soret bands at 420 and 370 nm is observed, which we assign as arising from six-and five-coordinated heme, respectively. There is also a broad absorption envelope at 500-600 nm and a ligand-to-metal charge transfer band at 650 nm ( Fig. 2A). These bands are similar to those reported for heme bound to proteins through Cys, Cys/His or Cys/X coordination (Table S1 (8)). The presence of two bands in the Soret region is indicative of heterogeneity, which may arise either from changes in axial ligands or from different orientations of the bound heme (8)(9)(10). The ligands to the heme in hERG3 were not identified. The affinity of ferric heme for hERG3eag was determined by titration (Kd = 7.02 ± 0.35 μΜ, Fig. 2A, inset top). Heme affinity can also be extracted from the first-order rate constant for transfer of heme to apo-myoglobin (which has a very high affinity for heme (11)); we measured this rate constant as koff = 0.03 s -1 ( Fig. 2A, inset bottom), which is several orders of magnitude higher than for the globins and more in the range observed for other regulatory heme proteins (12).

Spectroscopic analysis of heme binding.
Resonance Raman spectroscopy was used to provide more quantitative insight into the interaction of hERG3-eag with heme. In the highfrequency region, the spectrum of free heme displays the distinctive features of a ferric fivecoordinate high-spin complex (5c-HS), with characteristic bands at 1372 (v4), 1490 (v3), and 1570 cm -1 (v2) (Fig. 2B(i)). The v10 mode of the 5c-HS species is hidden by the vinyl stretching bands expected in the 1610-to 1640 cm -1 region (1618 cm -1 for the in-plane and 1628 cm -1 for the out-ofplane vinyl group (13)). The ferric hERG3-eagheme complex shows significant differences from free heme (Fig. 2B(ii)). All the modes described for ferric five-coordinate high-spin complex (5c-HS) are present for the hERG3-eag-heme complex. Additionally, we observe bands at 1502 (v3), 1553 & 1585 cm -1 (v2), and 1640 cm -1 (v10). These modes indicate the presence of a sixcoordinate low-spin complex (6c-LS) (Table S1) (14). These resonance Raman data are in agreement with the absorption spectra above, and demonstrate the formation of a six-coordinated heme species with a specific binding environment. EPR analyses support these conclusions, as the spectrum of the hERG3-eag-heme complex shows the formation of a low-spin heme complex (Fig.  2C). The spectrum is indicative of a mixture of species and was best simulated with two components (see Fig. 2C). The g-values for both are in good agreement with those of other ferric heme proteins bearing cysteine coordinated opposite a neutral sixth ligand (8). A Cys/His ligation is favoured for both components as suggested by the placement in the Blumberg-Peisach diagram (Fig. 2C). Carbon monoxide (CO) binds tightly (Kd = 1.03 ± 0.37 μM, Fig. 3A) to the ferrous hERG3-eag-heme complex, and gives a species (Soret band at 420 nm; Q-bands at 540 and 569 nm) that is characteristic of a normal CO-bound heme species. An identical species (Soret band at 420 nm; Q-bands at 540 and 569 nm), is formed on reaction of apo-hERG3-eag domain with a preformed heme-CO complex, and with an affinity (Kd = 10.55 ± 1.34 μM, data not shown) that is within reasonable range of the Kd for binding of CO to the ferrous hERG3-eag complex (Kd = 1.03 ± 0.37 μM) as above. These spectra for the hERG3-heme-CO complex formed by two different routes are significantly different from that of a free heme-CO complex (lmax = 407, 537 and 567 nm (12)), which indicates formation of a specific protein-heme-CO complex. The CO ligand is easily dissociated (koff = 0.03 s -1 ) in the presence of nitric oxide (Soret band at 390 nm, Fig. 3C), which is characteristic of a fivecoordinated NO-bound heme protein complex (15,16). These data indicate that the hERG3-eagheme complex is competent for binding of biologically relevant gaseous ligands. Structure of the hERG3-eag domain. The purified hERG3-eag protein was crystallised in the apo-form, in a monomeric state. The structure of the PAS domain (residues 18-135) is shown in Fig. 4A. The hERG3-eag domain has a canonical PAS fold that comprises of five antiparallel βsheets flanked by 3 α-helices, forming a hydrophobic cleft. This cleft is formed between the inner face of the β-sheet and the conformationally mobile F-helix. It is conserved in PAS domains known to bind ligands, including heme (3). The hERG3-eag structure contains a Cys39-Cys64 disulphide bond (Fig. 4A) situated 12 residues from the F-helix, and 13 residues from the Cap domain. The role of the disulphide bond is currently unknown and was not tested in this work, but it may impart a redox sensing functionality to the PAS domain (as previously suggested (17)(18)(19)(20)). The first 17 residues at the N-terminus are not seen in interpretable electron density in the crystal; furthermore, residues 18-23 are of weak density, indicating heterogenetity in this region (the Nterminal Cap). Mass spectrometry data (Fig. S2C) show there is a mixture of molecular weights with varying degrees of truncation at the N-terminus ( Fig. S2D), and the observed density most likely reflects this mixture. As a significant proportion of the protein is unaccounted for in the model, this probably explains the relatively high R values for a structure at this resolution (see Table S2). Despite attempts to obtain a co-crystal structure of heme-bound hERG3-eag, extensive cocrystallisation and soaking experiments were unsuccessful. As we have noted previously (6), this does not mean that heme cannot bind to the protein, as the spectroscopic data clearly demonstrate that it does. It is more likely that the particular conformation selected for in the crystal structure is incompatible with heme binding, so that heme cannot bind to the protein during the crystallisation process. Structural flexibility of the protein that is linked to the heme binding event (see Discussion) might also affect whether heme co-crytallises with the protein.

Effect of heme on hERG3 channels.
To measure the functional response of hERG3 channels to heme, hERG3 was heterologously expressed in CHO cells. In whole-cell recordings, K + currents were elicited with the characteristic features of the hERG family (Fig. 5A), including slow voltage dependent activation and deactivation, rapid onset and recovery from inactivation and inhibition by astemizole (data not shown). Application of heme ( Fig. 5B-E) caused a substantial inhibition of hERG3 current at all potentials (shown here after >3 mins). Mean (± SEM) heme inhibition of peak tail currents with a test potential to +40 mV was 63 ± 12 % (p<0.05, n = 3). Heme caused a small but significant negative shift in the voltage for half maximal activation of 6.7 % (p<0.05, n = 3) (Fig.  5E). The lipophilic properties of heme and its ability to interact with membrane phospholipids allows it to be transported across the plasma membrane via passive diffusion (21). To test if addition of intracellular heme also inhibited hERG3 channels, currents were recorded using the inside-out configuration of the patch clamp technique and heme was applied directly to the cytoplasmic side of excised membrane patches. As reported with many other ionic currents, upon excision a rapid decrease of hERG3 currents was observed in inside-out macropatches, consistent with loss of channel activity (run-down) upon removing the channels from the cellular environment. Addition of PIP2 (10 µM) to the perfusate ameliorated run-down and resulted in stable recording conditions (Fig. 5G, H). Subsequent application of heme ( Fig. 5F-H) to the intracellular side of the membrane caused inhibition of hERG3 currents in a similar manner as extracellularly applied heme in whole cell recordings. The time courses of heme inhibition were comparable, suggesting factors other than membrane diffusion of heme determine rates of hERG3 current inhbition. . These data demonstrate that heme inhibits hERG3 channel activity, thus connecting the heme binding event to modulation of channel function.

DISCUSSION
The ether-à-go-go (EAG, Kv10-12) family of voltage gated K + channels are regulators of neuronal and cardiac cell action potential firing (excitability). In humans, the family comprises hEAG, hERG and hELK channels (22,23). In this work, we have identified heme-dependent regulation of hERG3 channel activity. Formation of a six-coordinated heme species is supported from resonance Raman data, as well as EPR and UV-visible spectra, and a heme-binding PAS domain within the eag-domain of the channel has been identified. Recent cryo-EM structural models of rat EAG1 and hERG1 confirm that the four eag-domains are arranged around the periphery of the tetrameric intracellular assembly formed by the cNBHD and C-linkers (24,25). The function of this large intracellular complex is to modulate voltage dependent gating; profoundly influencing the time course and amplitudes of potassium currents during physiological processes such as action potentials. It sits underneath the transmembrane domains, with the C-linker connecting directly with the S6 activation gate. The PAS domain makes contacts predominantly with the CNBHD, whereas the Cap-domain extends towards the interface between the voltage sensor, C-linker and channel pore and thus is perfectly positioned to influence voltage and time dependent gating properties. Interestingly, functional studies reveal that deletion of the PAS and Cap domains has differential effects on the gating properties of hERG1 and hEAG1, suggesting that specific contacts with gating machinery of the two channels are different (26,27) In hEAG1 channels, the eag-domain is also required for calciumcalmodulin dependent inhibition of current (26). hERG3 gating is similar to hERG1 and it is likely that the eag domain has a similar role in both channels, which is to slow deactivation gating and enhance inactivation gating. PAS domains, which are known to bind heme (4,5), have well established roles in regulating the function of the related hERG1 channel (7,28,29), but heme binding to this channel has not been established. Heme has been shown to potently modulate the activity of a range of other potassium ion channels such as Kv1.4, large conductance Ca 2+ -activated K channels and ATP activated K channels (12,(30)(31)(32)(33)(34)(35)(36). Our observation that heme binding reduces hERG3 activity furthers the notion that heme plays a deliberate modulatory role in these ion channel systems. As we noted in the introduction, the capacity for the heme group to also bind cell signalling gases (CO, NO, O2 or even H2S) vastly broadens the range of stimuli that these ion channels could potentially be modulated by. It is worth noting that heme is important for neuronal survival (37), and so a role for heme in the regulation of the 'neuronal' channels hERG2 and hERG3 might reasonably be anticipated. At present, the biological basis for an interaction of heme with hERG3 is unknown, but a significant portion of brain damage caused during haemorrhagic stroke is believed to be due to the longer lasting, cytotoxic effects of heme (38). More generally, an abundance of heme after traumatic events such as stroke and ischemia could conceivably disrupt the regulatory balance of ion channels in the brain and has cytotoxic implications for the cells involved (39). Heme dependent inhibition of hERG3 would reduce repolarising K + currents, potentially increasing neuronal excitability and susceptibility to cell death. An abundance of heme after traumatic events thus has the capacity to disrupt the dynamic processes surrounding the regulation of ion channel function if they are sensitive to heme. We did not identify the heme binding location in hERG3-eag. However, we have compared the structure of apo-hERG3-eag with those of other heme-binding PAS domain proteins (EcDOS (40) and FixL (41)). Overlay of hERG3 with those of EcDOS and FixL shows that, compared to the apo-hERG3 structure, there is a large movement of the F-helix in the heme-bound EcDOS and FixL structures to accommodate binding of the heme group, Fig. 4B, C. It is feasible, therefore, that a similar mode of heme binding might occur in hERG3 if its F-helix is similarly flexible. Relevant in this context is the disulphide bond between Cys39 and Cys64, which is located 12 residues from the F-helix via a flexible loop. It has been proposed that disulphide bridges might serve as redox sensors in cells, by switching between thiol and disulphide structures (17)(18)(19)(20). Loss of the Cys39-Cys64 disulphide could facilitate movement of the F-helix, allowing heme to bind to the protein. The ligands to the bound heme are not identified as yet but candidate residues are His70, His77, and Cys66 (Fig. 4A), which are in the vicinity of the proposed heme binding site based on the comparison with EcDOS and FixL. Cys/His axial ligations would be consistent with our spectroscopic data and with known ligations in other regulatory heme proteins (4,5). Based on our structure, we identify one further possible mechanism of heme-dependent regulation. The N-terminal Cap domain (residues 1-26) in hERG3-eag is mostly unresolved in our structure. In hERG1 the Cap domain, which contains a flexibly-linked, but stable amphipathic helix, is already known to affect channel activity (27,42,43). So in addition to affecting the conformation of the F-helix (as above), it is possible that heme binding to the PAS domain in hERG3 could also affect the orientation of the adjacent and potentially highly mobile Cap domain (Fig. 1). This N-terminus is oriented towards the pore in the structure of the channel (25), and movements of the Cap domain might affect the conformations of the nearby cyclic nucleotide-binding homology domain, C-linker, voltage sensing domain, or pore forming domains as shown Fig. 6. Structural adjustments of this kind, in response to heme binding, would provide a mechanism for closure of the channel, due to the proximity of the N-terminal Cap domain to vital regulatory domains of channel gating (25). Relevant in this context is the observation that free heme affects channel function in the A-type potassium channels and, crucially, that the heme binds to the inactivation domain on the N-terminal region (30). We have no evidence for heme binding directly to the Cap domain in hERG3-eag, but changes in conformation of the N-terminal regions -induced either directly (by heme binding to N-termini), or indirectly (by heme binding to adjacent domains, such as PAS) -might plausibly provide a mechanism for ion channel regulation.

EXPERIMENTAL PROCEDURES
Chemicals and reagents. All reagents were from Sigma-Aldrich (Dorset, England) unless otherwise stated. CO solutions were prepared by bubbling bath solution with CO gas. Iron protoporphyrin IX (hemin, Fig. S1A) was purchased from Sigma Aldrich. Aqueous stock solutions of heme for all spectroscopic and electrophysiological experiments were prepared by dissolving solid heme in 0.1 M NaOH; final concentrations were calculated spectrophotometrically (ε385 = 58.4 mM −1 cm −1 (44)) and diluted accordingly to 500 nM prior to use. Heme-agarose affinity purification and mass spectrometry of neuronal lysates. Neuronal lysates were screened for possible heme-binding proteins using a modification of a previously published heme-agarose affinity (45). Binding of target proteins to heme-agarose was performed as previously (46), with modifications, Fig. S1B, that minimise pulling down proteins that bind to the heme-agarose resin non-specifically. Full details are given in the supplementary information. Using this approach, only proteins that were displaced from the agarose beads to bind specifically to the (heme) competitor (i.e. more likely to be bona fide heme binding proteins) were identified and subjected to proteomics analysis, , 1 mg/ml lysozyme and 1mg/ml DNAase), lysed by sonication and centrifuged to remove cell debris. Cell lysates were loaded onto a 5 ml Ni 2+ -NTA (Qiagen) affinity column equilibrated with 50 mM phosphate/100 mM NaCl, pH 7.00, 10% (vol/vol) glycerol and 15 mM imidazole. After washing with equilibration buffer the His6/S-tagged proteins were eluted with 50 mM phosphate/100 mM NaCl, pH 7.0, 10% glycerol and 100 mM EDTA. The His6/S-tag was cleaved with a Histagged TEV-protease during overnight dialysis. The His6/S-tag and His-tagged TEV protease were removed by reverse Ni 2+ -NTA affinity column and proteins were purified to homogeneity with size exclusion chromatography (Superdex 200, GE Healthcare) using 50 mM phosphate/100 mM NaCl, pH 7.0, Fig. S2B. Protein concentrations were determined using a Bradford assay. Optical Absorption Spectroscopy. Absorption spectra (25.0 o C) were obtained using a double beam spectrophotometer (Perkin Elmer Lambda 40) or a Kontron Uvikon UV-vis spectrometer. Ferric heme-bound hERG3-eag was obtained by addition of heme to hERG3-eag in 50 mM HEPES, 50 mM NaCl, pH 7.5. The reduced protein was obtained by anaeorobic addition of dithionite, and the ferrous-CO complex by gentle bubbling of CO through the reduced protein.
Values of Kd for binding of ligands (heme, CO) to hERG3 (3-5 mM) were obtained at 25.0 o C in 50mM HEPES/50mM NaCl, pH 7.5 as described previously (12). Heme dissociation was measured by reacting the ferric heme-hERG3-eag complex (6.4 μM) with a 5.6-fold excess of apo-myoglobin (36 μM); the reaction was followed at 408 nm (50 mM HEPES/50 mM NaCl pH 7.5). Resonance Raman Spectroscopy. Samples (50 μL, in 50 mM HEPES, 50 mM NaCl, pH 7.5) of ferric heme and its complexes were prepared in quartz EPR tubes and placed in a homemade spinning cell, at room temperature, to avoid local heating and to prevent photo-dissociation and degradation. Protein concentrations were in the range 90-120 μM, and sub-stoiochiometric concentrations of heme (to 0.8 equivalents) were added to the protein to ensure that no excess of heme was present. Raman excitation at 413.1 nm was achieved with a laser power <10 mW from an Innova 90 Kr + laser (Coherent, Palo Alto, California). Resonance Raman spectra were recorded using a two-stage monochromator (U1000, Jobin Yvon, Longjumeau, France) equipped with a front illuminated, deep-depleted CCD detector (Jobin Yvon, Longjumeau, France). Spectra correspond to an average of three different 1 h accumulations. The spectral accuracy was estimated to be ±1 cm -1 . Baseline correction was performed using GRAMS 32 (Galactic Industries, Salem, NH) and Origin®. Sample integrity was verified by following resonance Raman spectral evolution during the experiment. EPR spectroscopy. EPR spectra were recorded on an Elexsys 500 X-band spectrometer (Bruker) equipped with a continuous-flow ESR 900 cryostat and an ITC504 temperature controller (Oxford Instruments, Abingdon, UK). Simulations were performed by using the Easyspin software package (47) and routines written in the Dorlet laboratory. Samples (100 μl, 100 μM total heme concentration, 4-to 5-fold excess protein) of the ferric heme complexes of hERG3-eag were prepared in 50 mM HEPES and 50 mM NaCl, pH 7.5, and transferred to quartz EPR tubes. Electrophysiology. hERG3 currents were expressed in Chinese hamster ovary (CHO) cells and currents were recorded using either wholecell or macropatch (inside-out or cell attached) configurations of the patch clamp technique. hERG3 currents were recorded with an Axopatch 200 voltage clamp amplifier, and digitized using a Digidata 1440A interface, and acquired and analysed using pClamp 10 (Table S2). Data were processed and scaled with DIALS software from the CCP4 program suite (48). The structure was determined by molecular replacement in PHASER (49) using the hERG1 PAS domain [PDB 4HQA (50)] as the search model and refined with REFMAC5 (51). The model was refined to a resolution of 1.39 Å with Rwork and Rfree values of 18.2% and 23.4%, respectively, the final cycles of refinement included extrapolated hydrogen atoms with anisotropic atomic displacement parameters (B) for all atoms. Data collection and refinement statistics are in Table S2 (PDB ID 6Y7Q) and the weighting scheme is given in Table S3. Mass spectrometry analysis was carried out on a hERG3-eag crystal. The crystal was dissolved in 50 mM Hepes, 50 mM NaCl pH 7.5 and LC-MS was carried out using an RSLCnano HPLC system (Thermo Scientific) and an LTQ-Orbitrap-Velos mass spectrometer (Thermo Scientific). Samples were loaded at 0.1 mL/min onto a Vydac C8 5µm 250mm x 1mm I.D. reverse phase column (Grace Davison). The protein was desalted for 10 minutes in the loading buffer (0.1% formic acid) before elution using a 10 minute linear gradient from 3-96% B (80% acetonitrile / 0.1% formic acid). The output of the column was sprayed directly into the H-ESI2 electrospray ion source of the mass spectrometer maintained at 5kV. The FT analyser was set to acquire 10 microscans over the m/z range 800-2000 Da in positive ion mode. The maximum injection time for MS was 150 ms and the AGC target setting was 3e 6 Figure 1. Schematic of the hERG3 channel subunit. hERG, which is part of the EAG family of ion channels, has four subunits which assemble to form a tetrameric structure (left). Each subunit contains a transmembrane region as well as cytoplasmic N-and C-terminal regions. The transmembrane region contains voltage sensor (helices S1-S4, dark grey) and pore-forming (helices S5-S6, magenta) domains inside the membrane bilayer. The C-terminal cytoplasmic region contains a cyclic nucleotide-binding homology domain (CNBHD, red), which is connected to the transmembrane region via a structured domain usually referred to as the C-linker. The N-terminal cytoplasmic region (approximately 400 residues) contains a region that is known as the eag domain (residues 1-135), which itself comprises a Cap domain (residues 1-25, light blue) and a PAS domain (residues 26-135, purple). This eag-domain fragment (residues 1-135) has been expressed in this work and is referred to as hERG3-eag in this paper. Absorbance changes at 408 nm on binding of the ferric hERG3-eag complex to apo-myoglobin. Data were fitted to the first order decay process, yielding k obs = 0.03 s -1 . (B) Room temperature high-frequency resonance Raman spectra of (i) free heme, (ii) the ferric hERG3-eag-heme complex. All spectra were collected with 413.1 nm laser excitation. (C) Left: X-band EPR spectrum (black trace) of the ferric hERG3-eag-heme complex in the low-spin region along with the simulated spectrum (red trace). Experimental conditions: microwave frequency, 9.38 GHz; microwave power, 0.064 mW; field modulation amplitude, 2 mT; field modulation frequency, 100 kHz; temperature, 15 K; [heme] = 100 μM, 5-fold excess of protein in HEPES buffer 50 mM, pH 7.5, NaCl 50 mM. Simulation parameters : species 1 (75%) g-values (g-strain) g z1 = 2.42 (0.06) g y1 = 2.27 (0.00) g x1 = 1.91 (0.04); species 2 (25%) g-values (g-strain) g z2 = 2.50 (0,07) g y2 = 2.28 (0.00) g x2 = 1.90 (0.06); Lorentzian linewidth full-width at half-maximum 4mT. Right: Blumberg-Peisach correlation diagram showing EPR parameters plotted for various heme proteins. . Inset: the 421 nm time course, fitted to a threeexponential process; using the dominant (50%) phase a rate constant for dissociation of CO, k off(CO) , was determined (k off(CO) = 0.03 s -1 ).