The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR.

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

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.

Measurement of Interpeptidic CuII Exchange Rate Constants of CuII-Amyloid-ß Complexes to Small Peptide Motifs by Tryptophan Fluorescence Quenching.

The interpeptidic CuII exchange rate constants were measured for two Cu amyloid-ß complexes, Cu(Aß1-16) and Cu(Aß1-28), to fluorescent peptides GHW and DAHW using a quantitative tryptophan fluorescence quenching methodology. The second-order rate constants were determined at three pH values (6.8, 7.4, and 8.7) important to the two Cu(Aß) coordination complexes, components Cu(Aß)I and Cu(Aß)II. The interpeptidic CuII exchange rate constant is approximately 104 M-1 s-1 but varies in magnitude depending on many variables. These include pH, length of the Aß peptide, location of the anchoring histidine ligand in the fluorescent peptide, number of amide deprotonations required in the tryptophan peptide to coordinate CuII, and interconversion between Cu(Aß)I and Cu(Aß)II. We also present EPR data probing the CuII exchange between peptides and the formation of ternary species between Cu(Aß) and GHW. As the nonfluorescent GHK and DAHK peptides are important motifs found in the blood and serum, their ability to sequester CuII ions from Cu(Aß) complexes may be relevant for the metal homeostasis and its implication in Alzheimer's disease. Thus, their kinetic CuII interpeptidic exchange rate constants are important chemical rate constants that can help elucidate the complex CuII trafficking puzzle in the synaptic cleft.

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study.

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.

A heme-binding domain controls regulation of ATP-dependent potassium channels.

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Revisiting the Val/Ile Mutation in Mammalian and Bacterial Nitric Oxide Synthases: A Spectroscopic and Kinetic Study.

Nitric oxide is produced in mammals by the nitric oxide synthase (NOS) isoforms at a catalytic site comprising a heme associated with a biopterin cofactor. Through genome sequencing, proteins that are highly homologous to the oxygenase domain of NOSs have been identified, in particular in bacteria. The active site is highly conserved except for a valine residue in the distal pocket that is replaced with an isoleucine in bacteria. This switch was previously reported to influence the kinetics of the reaction. We have used the V346I mutant of the mouse inducible NOS (iNOS) as well as the I224V mutant of the NOS from Bacillus subtilis (bsNOS) to study their spectroscopic signatures in solution and look for potential structural differences compared to their respective wild types. Both mutants seem destabilized in the absence of substrate and cofactor. When both substrate and cofactor are present, small differences can be detected with Nω-hydroxy-l-arginine compared to arginine, which is likely due to the differences in the hydrogen bonding network of the distal pocket. Stopped-flow experiments evidence significant changes in the kinetics of the reaction due to the mutation as was already known. We found these effects particularly marked for iNOS. On the basis of these results, we performed rapid freeze-quench experiments to trap the biopterin radical and found the same results that we had obtained for the wild types. Despite differences in kinetics, a radical could be trapped in both steps for the iNOS mutant but only for the first step in the mutant of bsNOS. This strengthens the hypothesis that mammalian and bacterial NOSs may have a different mechanism during the second catalytic step.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.

Structural Insights into the Nature of Fe0 and FeI Low-Valent Species Obtained upon the Reduction of Iron Salts by Aryl Grignard Reagents.

Mechanistic studies of the reduction of FeIII and FeII salts by aryl Grignard reagents in toluene/tetrahydrofuran mixtures in the absence of a supporting ligand, as well as structural insights regarding the nature of the low-valent iron species obtained at the end of this reduction process, are reported. It is shown that several reduction pathways can be followed, depending on the starting iron precursor. We demonstrate, moreover, that these pathways lead to a mixture of Fe0 and FeI complexes regardless of the nature of the precursor. Mössbauer and 1H NMR spectroscopies suggest that diamagnetic 16-electron bisarene complexes such as (η4-C6H5Me)2Fe0 can be formed as major species (85% of the overall iron quantity). The formation of a η6-arene-ligated low-spin FeI complex as a minor species (accounting for ca. 15% of the overall iron quantity) is attested by Mössbauer spectroscopy, as well as by continuous-wave electron paramagnetic resonance (EPR) and pulsed-EPR (HYSCORE) spectroscopies. The nature of the FeI coordination sphere is discussed by means of isotopic labeling experiments and density functional theory calculations. It is shown that the most likely low-spin FeI candidate obtained in these systems is a diphenylarene-stabilized species [(η6-C6H5Me)FeIPh2]- exhibiting an idealized C2v topology. This enlightens the nature of the lowest valence states accommodated by iron during the reduction of FeIII and FeII salts by aryl Grignard reagents in the absence of any additional coligand, which so far remained rather unknown. The reactivity of these low-valent FeI and Fe0 complexes in aryl-heteroaryl Kumada cross-coupling conditions has also been investigated, and it is shown that the zerovalent Fe0 species can be used efficiently as a precursor in this reaction, whereas the FeI oxidation state does not exhibit any reactivity.

Remote His50 Acts as a Coordination Switch in the High-Affinity N-Terminal Centered Copper(II) Site of α-Synuclein.

Parkinson's disease (PD) etiology is closely linked to the aggregation of α-synuclein (αS). Copper(II) ions can bind to αS and may impact its aggregation propensity. As a consequence, deciphering the exact mode of Cu(II) binding to αS is important in the PD context. Several previous reports have shown some discrepancies in the description of the main Cu(II) site in αS, which are resolved here by a new scenario. Three Cu(II) species can be encountered, depending on the pH and the Cu:αS ratio. At low pH, Cu(II) is bound to the N-terminal part of the protein by the N-terminal amine, the adjacent deprotonated amide group of the Asp2 residue, and the carboxylate group from the side chain of the same Asp2. At pH 7.4, the imidazole group of remote His50 occupies the fourth labile equatorial position of the previous site. At high Cu(II):αS ratio (>1), His50 leaves the coordination sphere of the first Cu site centered at the N-terminus, because a second weak affinity site centered on His50 is now filled with Cu(II). In this new scheme, the remote His plays the role of a molecular switch and it can be anticipated that the binding of the remote His to the Cu(II) ion can induce different folding of the αS protein, having various aggregation propensity.

Synthesis, characterization and biological activity of Cu(II), Zn(II) and Re(I) complexes derived from S-benzyldithiocarbazate and 3-acetylcoumarin.

Cu(II), Zn(II) and Re(I) complexes have been synthesized with the Schiff base, N'-[1-(2-oxo-2H-chromen-3-yl)-ethylidene]-hydrazinecarbodithioic acid benzyl ester (SBCM-H) which was prepared by condensation of S-benzyldithiocarbazate and 3-acetylcoumarin. The metal complexes were characterized on the basis of various physico-chemical and spectroscopic techniques including elemental analysis and electrochemical studies, and FT-IR, UV-Vis, NMR, EPR and mass spectroscopy. The Schiff base was found to behave as a bidentate ligand coordinating with Cu(II) and Zn(II) in the thiolate form with 1:2 metal to ligand stoichiometry. Crystals suitable for X-ray diffractometry (XRD) were obtained from the reaction of ReCl(CO)5 with SBCM-H forming a centrosymmetric dimeric complex Re2L2(CO)6 linked by Re-S-Re bridges, where S is the thiolate sulfur of the N,S-bidentate ligand. This Re(I) complex is the first metal carbonyl complex with a bidentate dithiocarbazate ligand to have been characterized by XRD. Cytotoxicity assays revealed enhancement of the bioactivity of SBCM-H upon complexation. Both Cu(II) and Re(I) complexes are found to be active against human breast adenocarcinoma cancer cell lines MDA-MB-231 and MCF-7. TOC diagram.

Conjugation of a new series of dithiocarbazate Schiff base Copper(II) complexes with vectors selected to enhance antibacterial activity.

A new series of six Schiff bases derived from S-methyldithiocarbazate (SMDTC) and S-benzyldithiocarbazate (SBDTC) with methyl levulinate (SMML, SBML), levulinic acid (SMLA, SBLA), and 4-carboxybenzaldehyde (SM4CB, SB4CB) were reacted with copper(II), producing complexes of general formula ML2 (M = Cu(II), L = ligand). All compounds were characterized using established physicochemical and spectroscopic methods. Crystal structures were determined for three Schiff bases (SMML, SBML, SBLA) and two Cu(II) complexes (Cu(SMML)2 and Cu(SMLA)2). In order to provide more insight into the behavior of the complexes in solution, electron paramagnetic resonance (EPR) and electrochemical experiments were performed. The parent ligands and their respective copper(II) complexes exhibited moderate antibacterial activity against both Gram-negative and Gram-positive bacteria. The most active ligand (SB4CB) and its analogous S-methyl derivative (SM4CB) were conjugated with various vector moieties: polyarginines (R1, R4, R9, and RW9), oligoethylene glycol (OEG), and an efflux pump blocker, phenylalanine-arginine-ß-naphthylamide (PAßN). Nonaarginine (R9) derivatives showed the most encouraging synergistic effects upon conjugation and complexation with copper ion including enhanced water solubility, bacteria cell membrane permeability, and bioactivity. These Cu(II)-R9 derivatives display remarkable antibacterial activity against a wide spectrum of bacteria and, in particular, are highly efficacious against Staphylococcus aureus with minimum inhibitory concentration (MIC) values of 0.5-1 µM. This pioneer study clearly indicates that the conjugation of cell-penetrating peptides (CPPs) to dithiocarbazate compounds greatly enhances their therapeutic potential.

The green cupredoxin CopI is a multicopper protein able to oxidize Cu(I).

Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

EPR characterisation of the ferrous nitrosyl complex formed within the oxygenase domain of NO synthase.

Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.

Bis(thiosemicarbazone) copper complexes: mechanism of intracellular accumulation.

The molecular basis of Alzheimer's disease has not been clearly established, but disruption of brain metal ion homeostasis, particularly copper and zinc, might be closely involved in the pathogenesis of this disease and its characteristic ß-amyloid neuropathological features. The use of complexes of copper with bis(thiosemicarbazones) ([Cu(btsc)]) has been proposed for the treatment of Alzheimer's disease. Their mode of action could involve modulation of the concentration of copper or zinc, and it has been suggested that the compounds can modulate the production of ß-amyloid peptide at the neuron level. Furthermore, it has been reported that [Cu(btsc)] complexes can be reduced inside the cells. However, to our knowledge the intracellular reduction of these compounds has never been demonstrated. Thus, the goal of our study was to increase understanding of the mechanism of intracellular accumulation of [Cu(btsc)] complexes. Our results reveal that the intracellular concentration of copper inside the cells is very high and that these compounds are not P-glycoprotein substrates. This protein is a key element of the low permeability properties of the blood-brain barrier. Furthermore, no intracellular reduction of cupric ions was detected. Finally, once inside the cells, the complexes undergo aggregation, strongly suggesting that aggregation of complexes is the driving force responsible for their intracellular accumulation.

Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase.

H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.

Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.