Heme Propionate Groups Research Articles

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

How Do Preorganized Electric Fields Function in Catalytic Cycles? The Case of the Enzyme Tyrosine Hydroxylase.

Nature has devised intrinsic electric fields (IEFs) that are engaged in electrostatic catalysis of enzymes. But, how does the IEF target its function in enzymes that involve several reaction steps in catalytic cycles? To decipher the impact of the IEF on the catalytic cycle of an enzyme system, we have performed molecular dynamics and quantum-mechanical/molecular-mechanical (QM/MM) simulations on tyrosine hydroxylase (TyrH). The catalytic cycle of TyrH involves two reaction stages: the activation of H2O2 to form the active species of compound I (Cpd I), in the first stage, and the Cpd I-mediated hydroxylation of l-tyrosine to l-DOPA, in the second stage. For the first stage, the QM/MM calculations show that a heme-propionate group functions as a base to catalyze the O-O heterolysis reaction. For the second stage, the study reveals that the reaction is initiated by the His88-mediated proton-coupled electron transfer followed by the oxygen atom transfer from compound II (Cpd II) to the l-Tyr substrate. Importantly, our calculations demonstrate that the IEF in TyrH is optimized to promote the O-O bond heterolysis that generates the active species of the enzyme, Cpd I. However, the same IEF slows down the subsequent aromatic hydroxylation. Thus, the IEF in the TyrH enzymes does not catalyze the product formation step, but will selectively boost one or more challenging steps in the catalytic cycle. These findings have general implications on O2/H2O2-dependent metalloenzymes, which can expand our understanding of how nature has used electric fields as "smart reagents" in modulating the catalytic reactivity.

ЭЛЕКТРОСТАТИЧЕСКОЕ ВЗАИМОДЕЙСТВИЕ ГЛОБИНОВ С ФОСФОЛИПИДНЫМИ МЕМБРАНАМИ

The present study aimed to analyze the role of electrostatic interactions contributing to the stability of the native conformations of the heme group in the structure of sperm whale myoglobin (SWMb), horse heart myoglobin (HHMb), hemoglobin I (HbI) from the botfly Gasterophilus intestinalis (giHbI) and monomeric and dimeric hemoglobins HbI and HbII from the mollusk Lucina pectinata (lpHbI and lpHbII) as well as investigate a possible reason of destabilization due to interaction with negatively charged phospholipid membranes. It was shown that the native conformation of the heme cavity in these globins, both in its proximal and distal sides, is sustained by a system of hydrogen bonds involving the proximal and distal protein residues, both heme propionic acid groups and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom outside or in the protoporphyrin plane, affecting the efficiency of ligand binding, while in the distal part of the heme cavity the hydrogen bond network formed with the participation of the distal protein residue (HisE7 in swMb, hhMb and giHbI, and GlnE7 in lpHbII) should, apparently, stabilize conformation where the protein is able to donate hydrogen to O2 ligand...

Novel X-ray sequences and crystal structures of Persian and Starry sturgeon methemoglobins: Highlighting the role of heme pocket waters in causing autoxidation

Although there is a high sequence similarity between mammalian and fish hemoglobin (Hb), the oxidation and heme loss rates can vary greatly between them such that fish Hbs oxidise much more rapidly than mammalian Hbs. There is to date no sequence or structural data for any sturgeon Hb to reveal the level of autoxidation in these fish. In this study, novel high resolution X-ray sequences and crystal structures of methemoglobin (Met-Hb) from two sturgeon fish including Persian sturgeon (Acipenser percisus) and Starry sturgeon (Acipenser stellatus) belonging to the Caspian sea has been determined. A comprehensive sequence and structure comparison between these sturgeon Met-Hbs and a number of non-sturgeon and normal and sickle cell anaemia human Hb in varying heme states has been carried out highlighting (i) the structural variability in the heme propionate groups; (ii) the existence of certain residues or their displacement and shift in the heme pocket allowing entry of water molecules into the heme pocket; (iii) the importance of the number of water molecules in the heme pocket; (iv) the hydrogen bonding between oxygens of A and D propionate groups and that of waters in the heme pocket; and (v) the role of heme binding waters causing oxidative stress and heme autoxidation.

Three phases in pH dependent heme abstraction from myoglobin

The extent of heme extraction from myoglobin (Mb) by methylethyl ketone is found to be pH dependent and show three distinct phases. Parallel investigations of the protein using resonance Raman (rR) and circular dichroism (CD) across these pH regions indicate that these phases correspond to three different protonation steps in holoMb as the pH of the solution changed. The first transition occurs between pH5–6 and is due to the protonation of one of the heme propionate groups which disrupts its H-bonding with Arg 45 in the loop. The 2nd phase (pH5–4) likely involves the protonation of the 2nd propionate which H-bonds to Ser 92 in the F-helix. The third phase (pH<3.5) involves dissociation of the FeIIHis bond which eventually leads to complete heme dissociation and unfolding.

Electron transfer pathways in a multiheme cytochrome MtrF

In MtrF, an outer-membrane multiheme cytochrome, the 10 heme groups are arranged in heme binding domains II and IV along the pseudo-C2 axis, forming the electron transfer (ET) pathways. Previous reports based on molecular dynamics simulations showed that the redox potential (Em) values for the heme pairs located in symmetrical positions in domains II and IV were similar, forming bidirectional ET pathways [Breuer M, Zarzycki P, Blumberger J, Rosso KM (2012) J Am Chem Soc 134(24):9868-9871]. Here, we present the Em values of the 10 hemes in MtrF, solving the linear Poisson-Boltzmann equation and considering the protonation states of all titratable residues and heme propionic groups. In contrast to previous studies, the Em values indicated that the ET is more likely to be downhill from domain IV to II because of localization of acidic residues in domain IV. Reduction of hemes in MtrF lowered the Em values, resulting in switching to alternative downhill ET pathways that extended to the flavin binding sites. These findings present an explanation of how MtrF serves as an electron donor to extracellular substrates.

Effects of Azole Drugs on Conformational Dynamics and Energetics Associated with Diatomic Ligand Photorelease and Migration within Bacterial Flavohemoglobins

Bacterial flavohemoglobins (fHbs) are a widely distributed class of proteins which are though to protect microbes from nitrosative stress due to the immune response of a host organism via nitric oxide dioxygenase (NOD) activity. There has been interest lately in developing fHbs as targets for antibiotics due to their ubiquity in bacteria and the discovery of inhibition of NOD activity of fHbs by azole-based drugs which bind with high affinity (<12 μM). To determine how azole drugs affect fHbs we have used photoacoustic calorimetry (PAC) and transient absorption spectroscopy (TA) to calculate enthalpy and volume changes (ΔH and ΔV) and kinetic parameters for CO photorelease and rebinding to fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus (HMPSa) in the presence and absence of the drugs ketoconazole and miconazole. We observe that binding of drugs to fHbs results in a more exothermic ΔH for CO photorelease and faster bimolecular rebinding of CO. We also use classical molecular dynamics (cMD) to characterize ligand migration pathways present in FHP. We have conducted cMD simulations of CO-FHP WT and FHP WT containing free CO molecules using a locally enhanced sampling (LES) algorithm. We have carried out a 10 ns simulation of CO-FHP WT and a 30 ns simulation of the LES-enabled CO:FHP WT. We observe dissociation of 18 of 20 LES-enhanced CO molecules from the distal heme pocket. The primary escape pathways are between the CD-loop and heme-propionate groups and through a pathway exiting the protein matrix near the N-terminal end of the A-helix. These pathways are close to the locations of ketoconazole and miconazole binding predicted by AutoDock Vina, suggesting that drug binding inhibits NOD activity by closing migration pathways for O2 and NO.

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.

マルチへムシトクロム蛋白質MtrFの電子移動経路

In MtrF, an outer-membrane multiheme cytochrome, the 10 heme groups are arranged in heme binding domains II and IV along the pseudo-C2 axis, forming the electron transfer (ET) pathways. Previous reports based on molecular dynamics simulations showed that the redox potential (Em) values for the heme pairs located in symmetrical positions in domains II and IV were similar, forming bidirectional ET pathways [Breuer M, Zarzycki P, Blumberger J, Rosso KM (2012) J Am Chem Soc 134(24):9868–9871]. Here, we present the Em values of the 10 hemes in MtrF, solving the linear Poisson–Boltzmann equation and considering the protonation states of all titratable residues and heme propionic groups. In contrast to previous studies, the Em values indicated that the ET is more likely to be downhill from domain IV to II because of localization of acidic residues in domain IV. Reduction of hemes in MtrF lowered the Em values, resulting in switching to alternative downhill ET pathways that extended to the flavin binding sites. These findings present an explanation of how MtrF serves as an electron donor to extracellular substrates.

Intermolecular Recognition of the Antimalarial Drug Chloroquine: A Quantum Theory of Atoms in Molecules–Density Functional Theory Investigation of the Hydrated Dihydrogen Phosphate Salt from the 103 K X-ray Structure

The relevant noncovalent interaction patterns responsible for intermolecular recognition of the antiplasmodial chloroquine (CQ) in its bioactive diprotonated form, CQH22+, are investigated. Chloroquine dihydrogen phosphate hydrated salt (P21/c) was crystallized by gel diffusion. A high-resolution single-crystal X-ray diffraction experiment was performed at 103(2) K, and a density functional theory model for the in-crystal electron density was derived, allowing the estimation of the interaction energies in relevant molecular pairs. H2PO4– ions form infinite chains parallel to the monoclinic axis, setting up strong NH···O charge-assisted hydrogen bonds (CAHBs) with CQH22+. Couples of facing protonated quinoline rings are packed in a π···π stacked arrangement, whose contribution to the interaction energy is very low in the crystal and completely overwhelmed by Coulomb repulsion between positive aromatic rings. This questions the ability of CQ in setting up similar stacking interactions with the positively charged Fe-protoporphyrin moiety of the heme substrate in solution. When the heme/CQ adduct incorporates a Fe–N coordinative bond, stronger π···π interactions are instead established due to the lacking of net electrostatic repulsions. Yet, CAHBs among the protonated tertiary amine of CQ and the propionate group of heme still provide the leading stabilizing effect. Implications on possible modifications/improvements of the CQ pharmacophore are discussed.

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.

Structural Basis for Heme Transport By Hmut in Corynebacterium Glutamicum

Heme can be used as an iron source by some pathogenic bacteria, which have the heme acquisition system(s) to uptake heme. The typical heme acquisition system comprises several heme-binding proteins and an ABC transporter. The ABC heme transporter, HmuT-HmuUV is conserved in Gram-positive and Gram-negative bacteria. In this heme transporter system, HmuT, HmuU, and HmuV act as a substrate (heme)-binding protein, a heme permease, and an ATP-binding protein, respectively. In this study, the crystal structure of the heme-binding protein HmuT (CgHmuT) from Gram-positive bacterium Corynebacterium glutamicum will be reported at 1.4 Å resolution. The overall structure of CgHmuT reveals a bi-lobed topology formed by an N- and C-terminal domain. A heme molecule coordinated by His141 and Tyr242 is bound in a cleft between the N- and C-terminal domains. Though the global fold of CgHmuT is similar to that of HmuT from Yersinia pestis (YpHmuT), the coordination structure of the heme is different from each other. While two heme molecules are bound to YpHmuT, CgHmuT binds a single heme molecule with His141 and Tyr242 as the axial ligands. Intriguingly, CgHmuT binds a heme with two different orientations. Though the previously reported heme-binding proteins have the interaction between proteins and heme propionate groups in addition to axial ligation to iron, such interactions with heme propionates are absent in CgHmuT. The presence of two different conformations in CgHmuT might be due to a lack of specific interactions between the heme and protein except for the axial ligations. EPR spectroscopy reveals the heme in CgHmuT is a mixture of high-spin and low-spin states, indicating one of the axial ligand of the heme is in equilibrium between the ligated and unligated states. EPR spectra of wild-type, H141A-, and Y242A-CgHmuT suggest His141 will be reversibly dissociated/associated from/to the heme. The equilibrium between the six- and five-coordinate states in CgHmuT would be responsible for the regulation of heme-binding affinity to control the heme transport reaction between CgHmuT and HmuUV. The crystal structures of H141A and Y242A variants have also been determined in this study. Iwill discuss the molecular mechanism of heme recognition and heme transport by CgHmuT based on these structures.

Structural and functional alterations of myoglobin by glucose-protein interactions

The interaction of blood glucose with heme proteins plays a key role in inducing diabetes, a serious disease threatening human health. In this study, we investigated the non-covalent interaction between glucose and myoglobin (Mb), both theoretically and experimentally, using molecular dynamics (MD) simulation combined with spectroscopic studies. It revealed that glucoses can occupy the side pocket of Mb, and bind closely to one of the xenon cavities in Mb, by hydrogen bonding interactions with two propionate groups of heme as well as surrounding amino acids. These interactions alter the conformation of the heme active site slightly and lead to an enhanced peroxidase activity of Mb, as determined by kinetic studies. This study provides general information for glucose-heme proteins interactions, and also for blood glucose-protein interactions for patients with diabetes.

Heme-Transfer Mechanism of Structurally Similar Isd NEAT Domains of Staphylococcus Aureus Exhibiting Different Affinities for Heme

Near Transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins play an important role in importing heme from host animals to Staphylococcus aureus. IsdH, IsdA, and IsdC are anchored to the cell wall and display one or more copies of NEAT domain. The order of transfer of heme between the NEAT domains is as follows: IsdH-NEAT3 → IsdA-NEAT → IsdC-NEAT, though the three-dimensional structures are quite similar. In this study, we measured the difference of the affinities for heme between these NEAT domains and found that the affinity gradually increased in the downstream receptors. To gain insight into the atomistic mechanism for the difference, we performed in silico MD simulation and in vitro site-directed mutagenesis. The simulations showed that a cluster of negatively charged residues in the first loop between strand β1b and the 310 helix (loop 1 region) adversely influences the interaction with the propionate group of heme. Meanwhile, the higher affinity of IsdC was partly attributed to an internal salt-bridge formed between Glu88/Arg100 on binding to heme. On the other hand, we also observed that Phe130 of IsdC makes the β7-β8 hairpin region less flexible in the ligand-free form, which leads to reducing the entropy loss on binding to heme. Based on these calculations, we confirmed that substitution of these key residues decreased its affinity for heme. Importantly, these affinity-decreased IsdC mutants did transfer heme back to IsdA. Moreover, substitution of the loop 1 region in IsdH-NEAT3 for IsdC-type residues reversibly increased its affinity for heme and did not transfer to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold so that they exhibit different affinities for heme and promote the efficient transfer of heme.

Heme Binding Mechanism of Structurally Similar Iron-Regulated Surface Determinant Near Transporter Domains ofStaphylococcus aureusExhibiting Different Affinities for Heme

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7-β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

Redox-Controlled Proton Gating in Bovine Cytochrome c Oxidase

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method, we have identified two critical elements of the proton pump: a proton loading site near the propionate groups of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a postulated molecular model describing a detailed mechanism by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a 3, associated with the oxygen chemistry occurring in the heme a 3 site, during enzymatic turnover.

Effects of mutating aromatic surface residues of the heme domain of human sulfite oxidase on its heme midpoint potential, intramolecular electron transfer, and steady-state kinetics

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

Conformational Dynamics in Human Neuroglobin: Effect of His64, Val68, and Cys120 on Ligand Migration

Neuroglobin belongs to the family of hexacoordinate hemoglobins and has been implicated in the protection of neuronal tissue under hypoxic and ischemic conditions. Here we present transient absorption and photoacoustic calorimetry studies of CO photodissociation and bimolecular rebinding to neuroglobin focusing on the ligand migration process and the role of distal pocket residues (His64 and Val68) and two Cys residues (Cys55 and Cys120). Our results indicate that His64 has a minor impact on the migration of CO between the distal heme pocket and protein exterior, whereas the Val68 side chain regulates the transition of the photodissociated ligand between the distal pocket and internal hydrophobic cavities, which is evident from the increased geminate quantum yield in this mutated protein (Φ(gem) = 0.32 for WT and His64Gln, and Φ(gem) = 0.85 for Val68Phe). The interface between helix G and the A-B loop provides an escape pathway for the photodissociated ligand, which is evident from a decrease in the reaction enthalpy for the transition between the CO-bound hNgb and five-coordinate hNgb in the Cys120Ser mutant (ΔH = -3 ± 4 kcal mol(-1)) compared to that of the WT protein (ΔH = 20 ± 4 kcal mol(-1)). The extensive electrostatic/hydrogen binding network that includes heme propionate groups, Lys67, His64, and Tyr44 not only restricts the heme binding but also modulates the energetics of binding of CO to the five-coordinate hNgb as substitution of His64 with Gln leads to an endothermic association of CO with the five-coordinate hNgb (ΔH = 6 ± 3 kcal mol(-1)).

Redox-Controlled Proton Gating in Bovine Cytochrome cOxidase

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method, we have identified two critical elements of the proton pump: a proton loading site near the propionate groups of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a postulated molecular model describing a detailed mechanism by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a3, associated with the oxygen chemistry occurring in the heme a3 site, during enzymatic turnover.

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine–FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.