Ferric Heme Iron Research Articles

Applications of the Newly Developed Force-Field Parameters Uncover a Dynamic Nature of Ω-Loop C in the Lys-Ligated Alkaline Form of Cytochrome c.

Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso-1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6Å resolution) reveals a helical cap domain appended to the ⍺/β core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F.nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Heme protonation affects iron-NO binding in the NO transport protein nitrophorin

The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm−1, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.

Structural Insights on the Conversion of Cytochrome P450 to P420.

A characteristic feature of cytochromes P450* is that the complex formed between the ferrous heme iron and carbon monoxide generates an intense absorption band at 450 nm. This unique feature of P450s is due to the proximal thiolate Cys ligand coordinated to the heme iron. Various harsh treatments shift this band to 420 nm, thereby giving P420 which is most often associated with an inactive form of the enzyme. Various explanations have been put forward to explain the P450-to-P420 change ranging from protonation of the Cys heme ligand, displacement of the Cys ligand, or replacement of the Cys ligand with His. There are two crystal structures of the well-studied cytochrome P450cam that have a high fraction of P420. In one, P450cam is cross-linked to its redox partner, putidaredoxin (Pdx), and the second is P450cam crystallized in the absence of substrate. In both of these structures, a significant part of the substrate pocket is disordered and the poor quality of the electron density for the substrate indicates substantial disorder. However, in both structures there is no detectable change in the Cys-iron ligation or surrounding structure. These results indicate that the P450-to-P420 switch is due primarily to an opening and disordering around the substrate binding pocket and not ligand displacement or ligand swapping. Since it remains a possibility that ligand swapping could be responsible for P420 in some cases, we mutated to Gln the 3 His residues (352, 355, and 361) close enough to the proximal side of the heme that could possibly serve as heme ligands. The triple variant forms P420 which indicates that swapping Cys for His is not a requirement for the P450-to-P420 switch.

Hemoglobin Oxidation Reactions in Stored Blood.

Hemoglobin (Hb) inside and outside the red blood cells (RBCs) undergoes constant transformation to an oxidized form in a process known as autoxidation. The ferrous heme iron (Fe2+) of the prosthetic group is spontaneously transformed into an oxidized ferric (Fe3+) form, but under oxidative stress conditions a higher oxidation ferryl heme (Fe4+) is also formed. Although Fe3+ is a non-functional form of Hb, the Fe4+ is also extremely reactive towards other biological molecules due to its high redox potential. The RBC contains an effective reductive machinery that maintains Hb in the functional form with little oxidation during its life span. The redox transformation of Hb occurs to a lesser extent in young RBCs; it may, however, have detrimental effects on the integrity of these cells during ex vivo storage or when RBCs are subjected to pathogen reduction processes. In this review, Hb oxidation reactions (“oxidative lesion”) will be described, including details of how these reactions might impact the clinical use of stored or processed blood for therapeutic purposes.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Iron Acquisition in Mycobacterium tuberculosis.

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.

Spectroscopic evidence supporting neutral thiol ligation to ferrous heme iron.

The binding of neutral thiol (ethanethiol, EtSH) or thioether (tetrahydrothiophene, THT) to two types of heme proteins in their ferrous state has been investigated with UV-visible (UV-Vis) absorption and magnetic circular dichroism spectroscopy. For the second GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain from the sensory kinase MsmS (sGAF2), stepwise additions of these respective two sulfur-donor ligands to its dithionite-reduced ferrous form generate homogeneous six-coordinate low-spin ferrous complexes at both pHs 7.0 and 5.4. Similar complexes were partially formed for deoxyferrous soybean leghemoglobin with EtSH or THT within their solubility limits in water. The titrations cause significant UV-Vis spectra changes attributable to a five-coordinate to six-coordinate heme iron coordination change. For sGAF2, the resulting spectra are essentially identical for the both ligands, clearly indicating the direct binding of neutral thiol/thioether to ferrous heme iron as the distal ligand. On the other hand, the thiol EtSH binds to ferric sGAF2 in the anionic thiolate form, while thioether THT forms its ferric sGAF2 complex as a neutral ligand. These observations provide compelling evidence that neutral cysteine is a plausible ligand for ferrous heme proteins.

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

BackgroundThe nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1–4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. MethodsWe inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. ResultsRecombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between −65 and −100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. ConclusionsThree single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. General significancePrimary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.

Naturally Occurring Disease-Related Mutations in the 40-57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization.

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p Ktrigger). A second deprotonation stabilizes a pentacoordinate heme form (p Ka2). In this study, site-directed variants at the 41 and 48 positions in the 40-57 Ω-loop and at the 81 and 83 positions in the 71-85 Ω-loop reveal that conformational transitions in the 71-85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p Ktrigger and increase p Ka2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength.

Ligation and Reactivity of Methionine-Oxidized Cytochrome c.

Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein, but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO, implying the redox-linked ligand switch in the modified protein. Binding studies with the model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (p Ka = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells.

Different Mechanisms of Catalytic Complex Formation in Two L-Tryptophan Processing Dioxygenases.

The human heme enzymes tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3 dioxygenase (hIDO) catalyze the initial step in L-tryptophan (L-Trp) catabolism, the insertion of dioxygen into L-Trp. Overexpression of these enzymes causes depletion of L-Trp and accumulation of metabolic products, and thereby contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. Understanding the assembly of the catalytically active, ternary enzyme-substrate-ligand complexes is not yet fully resolved, but an essential prerequisite for designing efficient and selective de novo inhibitors. Evidence is mounting that the ternary complex forms by sequential binding of ligand and substrate in a specific order. In hTDO, the apolar L-Trp binds first, decreasing active-site solvation and, as a result, reducing non-productive oxidation of the heme iron by the dioxygen ligand, which may leave the substrate bound to a ferric heme iron. In hIDO, by contrast, dioxygen must first coordinate to the heme iron because a bound substrate would occlude ligand access to the heme iron, so the ternary complex can no longer form. Consequently, faster association of L-Trp at high concentrations results in substrate inhibition. Here, we summarize our present knowledge of ternary complex formation in hTDO and hIDO and relate these findings to structural peculiarities of their active sites.

Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.

The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0′ ∼ −118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30’s ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.

Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN−) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8kJmol−1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2kJmol−1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.

Prolonging the shelf life of Lumbricus terrestris erythrocruorin for use as a novel blood substitute

Limitations associated with the storage of red blood cells have motivated the development of novel blood substitutes that are able to withstand long-term storage at elevated temperatures. The hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an attractive blood substitute candidate, since it is resistant to oxidation and aggregation during storage. Several factors were investigated to optimize the thermal and oxidative stability of LtEc during storage, including pH, antioxidant supplements, and deoxygenation. A strategy for the reduction of fully oxidized LtEc with antioxidants was also developed. Overall, LtEc was shown to have the highest thermal stability in Ringer’s Modified Lactate solution with 10 mM HEPES at pH 7.0. Deoxygenation of the LtEc was also shown to significantly reduce oxidation of the ferrous heme iron (e.g., %Fe2+ after 7 d at 37 °C = 75.7%). However, even in cases where oxidation does occur, the addition of 1.8 mM ascorbic acid (AA) was found to reduce 98.3% of the oxidized LtEc (37 μM heme). Most importantly, the oxygen transport properties of LtEc were unaffected by storage at high temperatures or oxidation followed by reduction with AA. These results show that LtEc can be stored at high temperatures (37 °C) without any significant loss of function.

Characterization of Ground State Electron Configurations of High-Spin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors

We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2-y2), and 5E, (dxy)(dxz)2(dyz)(dz2)(dx2-y2), states of the heme Fe(II), i.e., 5B2 ⇆ 5E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼-20 kJ mol-1 and ∼-60 J K-1 mol-1, respectively, for the thermal equilibrium. Thus, the 5E state is slightly dominant over the 5B2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.

Insight into a Rapid Heme Transfer Reaction between Near Transporter Domains of Staphylococcus Aureus: A Theoretical Study using QM/MM and MD Simulations

Iron is versatile in biological systems and required cofactor in the growth and activity of all living organisms. Some gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system across its thick cell wall to steal iron from human hemoglobin and heme. Recent studies have demonstrated that heme extracted from hemoglobin is rapidly transferred into cytoplasm through NEAr Transporter (NEAT) domain of the Isd system, despite the high affinity for each domain. These observations suggest the presence of an intermediate NEAT•heme•NEAT complex. In this study, we performed restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domains to the donor NEAT•heme complexes and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. The overall structures of the complex models were stably maintained during subsequent more than 900-ns unrestrained simulations. Although the hydrogen bond between a propionate group of heme and the acceptor domain was completely maintained, the hydrogen bond between the other propionate group of heme and the donor domain was broken, which may facilitate the transition of heme from the donor to the acceptor. Moreover, subsequent structural optimization using the QM/MM method showed that the two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex, only if they are deprotonated. Therefore, the heme-transfer reaction proceeds by protonation on the coordinating tyrosine residue. Thus, these theoretical studies provide an atomistic insight into the intermediate structure allowing rapid heme transfer between the NEAT domains.

Rapid Heme Transfer Reactions between NEAr Transporter Domains of Staphylococcus aureus: A Theoretical Study Using QM/MM and MD Simulations.

In vertebrates, most iron is present as heme or is chelated by proteins. Thus, Gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system that transports heme across thick cell walls into the cytoplasm. Recent studies have demonstrated that heme is rapidly transferred between the NEAr Transporter (NEAT) domains of the Isd system, despite its high affinity toward each domain, suggesting the presence of an intermediate NEAT•heme•NEAT complex. In the present study, we performed short restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domain to the donor NEAT•heme complex and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. After turning off the restraints, complex structures were stably maintained during subsequent unrestrained MD simulations, except for the hydrogen bond between the propionate group of the heme molecule and the donor NEAT domain, potentially facilitating the transition of heme from the donor to the acceptor. Subsequent structural optimization using the quantum mechanics/molecular mechanics (QM/MM) method showed that two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex only if they were deprotonated. Based on these results, we propose a reaction scheme for heme transfer between NEAT domains.

Nitric Oxide Regulation of Mitochondrial Processes: Commonality in Medical Disorders

The vital status of diverse classes of eukaryotic mitochondria is reflected by the high degree of evolutionary modification functionally linked to ongoing multifaceted organelle development. From this teleological perspective, a logistical enhancement of eukaryotic cellular energy requirements indicates a convergence of metabolic processes within the mitochondrial matrix for optimal synthesis of ATP from ADP and inorganic phosphate and necessitates an evolutionarily driven retrofit of the primordial endosymbiont bacterial plasma membrane into the inner mitochondrial membrane. The biochemical complexity of eukaryotic inner membrane electron transport complexes linked to temporally-defined, state-dependent, fluctuations in mitochondrial oxygen utilization is capable of generating deleterious reactive oxygen species. Within this functional context, an extensive neurochemical literature supports the role of the free radical gas nitric oxide (NO) as a key signaling molecule involved in the regulation of multiple aspects of mitochondrial respiration/oxidative phosphorylation. Importantly, the unique chemical properties of NO underlie its rapid metabolism in vivo within a mechanistic spectrum of small oxidative molecules, free and protein-bound thiol adducts, and reversible binding to ferrous heme iron centers. Recent compelling work has identified a medically relevant dual regulation pathway for mitochondrial NO expression mediated by traditionally characterized NO synthases (NOS) and by enzymatic reduction of available cellular nitrite pools by a diverse class of cytosolic and mitochondrial nitrite reductases. Accordingly, our short review presents selected medically-based discussion topics relating to multi-faceted NO regulation of mitochondrial functions in human health and disease states.