Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.     

Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism

The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.     

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins.     

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Transient resonance Raman spectroscopy shows unrelaxed heme following CO photodissociation from cytochrome-c peroxidase

The 7 ns 436 nm pulses of an H2-shifted YAG laser have been used to photolyze the CO adduct of cytochrome-c peroxidase and produce the resonance Raman spectrum of the photoproduct. A 3 cm-1 downshift, relative to the spectrum of reduced enzyme, was observed for the porphyrin C-N breathing mode, v4. The downshift diminishes with decreasing CO /protein ratio, implying, in conjunction with a recent study of CO binding, that the unrelaxed heme is associated with adduct having a tilted, H-bonded FeCO unit. The downshift is eliminated when the phosphate buffer concentration is increased from 0.01 to 0.1 M. It is proposed that the heme relaxation under study involves a transition between two conformations, B and A, differing in the disposition of the distal residues, and having different v4 frequencies for unligated Fe(II) heme. Conformation B allows H-bonding to bound CO, and is favored at high CO and phosphate concentrations, while conformation A, which is unfavorable to CO H-bonding, is favored at low CO and phosphate concentrations. The recently reported absence of unrelaxed frequencies in the 7 ns photo-product of the CO adduct of horseradish peroxidase has been confirmed, and is attributed to lower stability for conformation B and a smaller A - B v4 difference.     

Induction of ER stress protects gastric cancer cells against apoptosis induced by cisplatin and doxorubicin through activation of p38 MAPK

Porphyromonas gingivalis acquires heme through an outer-membrane heme transporter HmuR and heme-binding hemophore-like lipoprotein HmuY. Here, we compare binding of iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) to HmuY with that of iron(III) protoporphyrin IX (protoheme) and protoporphyrin IX (PPIX) using spectroscopic methods. In contrast to PPIX, mesoheme and deuteroheme enter the HmuY heme cavity and are coordinated by His134 and His166 residues in a fully analogous way to protoheme binding. However, in the case of deuteroheme two forms of HmuY–iron porphyrin complex were observed differing by a 180° rotation of porphyrin about the α-γ-meso-carbon axis. Since the use of porphyrins either as active photosensitizers or in combination with antibiotics may have therapeutic value for controlling bacterial growth in vivo, it is important to compare the binding of heme derivatives to HmuY.     

Nitric oxide interaction with insect nitrophorins and thoughts on the electron configuration of the {FeNO}6 complex

The nitrophorins are NO-carrying heme proteins that are found in the saliva of two species of blood-sucking insects, the kissing bug (Rhodnius prolixus) and the bedbug (Cimex lectularius). In both insects the NO is bound to the ferric form of the protein, which gives rise to Kds in the micromolar to nanomolar range, and thus upon injection of the saliva into the tissues of the victim the NO can dissociate to cause vasodilation and inhibition of platelet aggregation. The structures of the proteins from each of these insects are unique, and each has a large component of beta-sheet structure, which is unusual for heme proteins. While the Rhodnius nitrophorins increase the effectiveness of their NO-heme proteins by also binding histamine, secreted by the victim in response to the bite, to the heme, the Cimex nitrophorin does not bind histamine but rather binds two molecules of NO reversibly, one to the heme and the other to the cysteine thiolate which serves as the heme ligand in the absence of NO. This requires homolytic cleavage of the Fe-S-Cys bond, which produces an EPR-active Fe(II)-NO complex having the {FeNO}7 electron configuration. For the Rhodnius nitrophorins, the heme of the {FeNO}6 stable NO complex could have the limiting electron configurations Fe(III)-NO+ or Fe(II)-NO+. While vibrational spectroscopy suggests the latter and Mossbauer spectroscopy cannot differentiate between a purely diamagnetic Fe(II) center and a strongly antiferromagnetically coupled Fe(III)-NO* center, the strong ruffling of the heme (with alternate meso-carbons shifted significantly above and below the mean plane of the porphyrin, and concomitant shifts of the beta-pyrrole carbons above and below the mean plane of the porphyrin ring, to produce a very nonplanar porphyrin macrocycle) may suggest at least an important contribution of the latter. The strong ruffling would help to stabilize the (dxz, dyz)4(dxy)1 electron configuration of low-spin Fe(III) (but not low-spin Fe(II)), and the dxy orbital does not have correct symmetry for overlap with the half-filled pi* orbital of NO. This Fe(III)-NO* electron configuration would facilitate reversible dissociation of NO.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: NMR studies of their tertiary and quaternary structures

Diruthenium-substituted Ru-Fe hybrid hemoglobins (Hb) were synthesized by heme substitution from protoheme to ruthenium (II) carbonyldeuteroporphyrin in the alpha or beta subunits. As the carbon monoxide coordinated to ruthenium (II) is not released under physiological conditions, deoxygenated Ru-Fe hybrid derivatives [alpha(Fe)2 beta(Ru-CO)2 and alpha(Ru-CO)2 beta(Fe)2] can serve as models for half-liganded Hbs. On the basis of proton NMR spectra of hyperfine-shifted proton resonances, these Ru-Fe hybrid Hbs have only small structural changes in the heme environment of the partner subunits at low pH. The proton NMR spectra of the intersubunit hydrogen-bonded protons also showed that the quaternary structures of the two complementary hybrids both remain in the "T-like state" at low pH, suggesting that the T to R structural conversion is induced by ligation of the third ligand molecule. Marked conformational changes in the heme vicinity are observed at high pH only for alpha(Ru-CO)2 beta(Fe)2, and its quaternary structure is converted into the "R state"; the alpha(Fe)2 beta(Ru-CO)2 hybrid does not undergo this change. This implies that the free-energy difference between the two quaternary states is smaller in the alpha-liganded hybrid than in the beta-liganded one.     

The role of transferrin in heme transport.

Porphyrin accumulation by proliferating cells, e.g., those associated with cancers or wounds, tends to correlate with increased transferrin receptor density. To determine whether transferrin might be implicated in porphyrin transport, fluorescence and absorption spectroscopy were used to study the interaction of porphyrins with transferrin. A single high-affinity binding site for heme and other porphyrins (Kd approximately 20-25 nM) was detected by fluorescence spectroscopy. Difference spectroscopy revealed three additional heme-binding sites. These sites were distinct from the iron-binding sites: 1) Apotransferrin and diferric transferrin bound porphyrins with equal affinity; 2) 59Fe was not displaced from transferrin by porphyrins. Murine erythroleukemia cells incubated with [59Fe]hemin-[125I]transferrin internalized both labels concomitantly. Accumulation of [59Fe]hemin could be blocked by a 100-fold excess of diferric transferrin but not by apotransferrin. These results indicate that cells can internalize exogenous heme, and possibly porphyrins, bound to transferrin via its receptor.     

Air-stable,heme-like water-soluble iron(II) porphyrin: in situ preparation and characterization

Preparation of the water-soluble, kinetically labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)porphyrin, Fe(II)TPPS⁴⁻, has been realized in neutral or weakly acidic solutions containing acetate buffer. The buffer played a double role in these systems: it was used for both adjusting pH and, via formation of an acetato complex, trapping trace amounts of iron(III) ions, which would convert the iron(II) porphyrins to the corresponding iron(III) species. Fe(II)TPPS⁴⁻ proved to be stable in these solutions even after saturation with air or oxygen. In the absence of acetate ions, however, iron(II) ions play a catalytic role in the formation of iron(III) porphyrins. While the kinetically inert iron(III) porphyrin, Fe(III)TPPS³⁻, is a regular one with no emission and photoredox properties, the corresponding iron(II) porphyrin displays photoinduced features which are typical of sitting-atop complexes (redshifted Soret absorption and blueshifted emission and Q absorption bands, photoinduced porphyrin ligand-to-metal charge transfer, LMCT, reaction). In the photolysis of Fe(II)TPPS⁴⁻ the LMCT process is followed by detachment of the reduced metal center and an irreversible ring-opening of the porphyrin ligand, resulting in the degradation of the complex. Possible oxygen-binding ability of Fe(II)TPPS⁴⁻ (as a heme model) has been studied as well. Density functional theory calculations revealed that in solutions with high acetate concentration there is very little chance for iron(II) porpyrin to bind and release O₂, deviating from heme in a hydrophobic microenvironment in hemoglobin. In the presence of an iron(III)-trapping additive that is much less strongly coordinated to the iron(II) center than the acetate ion, Fe(II)TPPS⁴⁻ may function as a heme model.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.     

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.     

Heme A biosynthesis

Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Interaction of heme and heme-hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport

Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme-hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme-hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme-hemopexin interact with electrons from PMET. We therefore propose that heme and heme-hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.     

Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.