Heme Prosthetic Group Research Articles

In Vitro Assembly of Catalase

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.

Different electrochemical behavior of adult and fetal hemoglobin at ionic liquid-carbon nanotube nanocomposite

The direct electrochemistry of human adult and fetal hemoglobin on the surface of nanocomposite containing the carboxylated multi-walled carbon nanotubes (MWCNTs) and a hydrophilic room temperature ionic liquid (RTIL) has been reported. The voltammetric results showed that both types of hemoglobins entrapped in RTIL-MWCNTs nanocomposite displayed a pair of well defined, quasi-reversible redox peaks, corresponding to the Fe(III)/Fe(II) redox couple of heme prosthetic group, with the formal potentials of −0.234 and −0.232 V (vs. Ag/AgCl) for human adult and fetal hemoglobin, respectively. Immobilized human fetal hemoglobin on RTIL-MWCNTs nanocomposite relative to immobilized human adult hemoglobin showed slight differences in reversibility, electron transfer rate and protein-substrate affinity toward hydrogen peroxide.

The globin superfamily: functions in nitric oxide formation and decay

Globin proteins are ubiquitous in living organisms and carry out a variety of functions related to the ability of their prosthetic heme group to bind gaseous ligands, such as oxygen, nitric oxide (NO), and CO. Moreover, they catalyze important reactions with nitrogen oxide species, such as NO dioxygenation and nitrite reduction. The formation of NO from nitrite is a reaction catalyzed by globins that has received increasing attention due to its potential as a hypoxic NO signaling mechanism. In this review, we revisit the current knowledge about the role of globins in NO formation and its physiological implications.

Mechanistic Characterization of a 2-Thioxanthine Myeloperoxidase Inhibitor and Selectivity Assessment Utilizing Click Chemistry–Activity-Based Protein Profiling

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 10⁶ M⁻¹ s⁻¹, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A and utilizing click chemistry-activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.

Redox Transitions in an Electrolyte-Free Myoglobin Fluid

Redox responses associated with the heme prosthetic group in a myoglobin-polymer surfactant solvent-free liquid are investigated for the first time in the absence of an electrolyte solution. Cyclic voltammograms from the biofluid exhibit responses that are consistent with planar diffusion of mobile charges in the melt. Temperature-dependent dynamic electrochemical and rheological responses are rationalized in terms of the effective electron hopping rate between heme centers and the transport of intrinsic ionic species in the viscous protein liquid.

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (-145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.

Chronic activation of heme free soluble guanylate cyclase leads to cardio-renal protection in experimental hypertension

Background The cytoprotective NO/sGC/cGMP-signalling pathway is impaired under oxidative stress conditions as observed for cardiovascular diseases due to oxidation and subsequent loss of the prosthetic sGC heme group [1,2]. Thus, the pool of heme free sGC is increased under pathological conditions. The unique property of sGC activators such as cinaciguat (BAY 58—2667) to selectively activate heme free sGC allows to target the disease associated form of sGC. In this study a therapeutic effect of long-term activation of heme free sGC was investigated in an experimental model of salt sensitive hypertension.

Targeting native and apo-sGC

Soluble guanylate cyclase (sGC), the receptor for nitric oxide (NO), is one component of the nitric oxide (NO)-sGC-cGMP signaling pathway, which plays a key role in the cardiovascular system regulating smooth muscle relaxation and vasodilation and has been implicated in remodeling events in the heart and vasculature. Triggered by the binding endothelial-derived NO to the prosthetic heme group, sGC in smooth muscle cells converts GTP to the secondary messenger cGMP. Small molecule activators of sGC have been identified that either have the ability to activate the native heme-containing form of the enzyme (Heme-Dependent activators) or have the ability to activate to heme-free or oxidized form of the enzyme (Heme-Independent activators). The therapeutic potential of activators of sGC is illustrated by the successful clinical evaluation by Bayer of a Heme-dependent activator riociguat in pulmonary arterial hypertension [1]. The characterization of both heme-dependent and heme-independent activators with regard to their biochemical interactions with the enzyme as well as preclinical in vivo activity in rodent models of systemic and pulmonary hypertension will be presented.

Oxygen-sensitive mitochondrial accumulation of cystathionine β-synthase mediated by Lon protease

Oxygen-sensitive accumulation and degradation, two opposite but intrinsically linked events, of heme proteins in mitochondria affect mitochondrial functions, including bioenergetics and oxygen-sensing processes. Cystathionine β-synthase (CBS) contains a prosthetic heme group and catalyzes the production of hydrogen sulfide in mammalian cells. Here we show that CBS proteins were present in liver mitochondria at a low level under normoxia conditions. Ischemia/hypoxia increased the accumulation of CBS proteins in mitochondria. The normalization of oxygen partial pressure accelerated the degradation of CBS proteins. Lon protease, a major degradation enzyme in mitochondrial matrix, recognized and degraded mitochondrial CBS by specifically targeting at the oxygenated heme group of CBS proteins. The accumulation of CBS in mitochondria increased hydrogen sulfide production, which prevented Ca(2+)-mediated cytochrome c release from mitochondria and decreased reactive oxygen species generation. Mitochondrial accumulation of heme oxygenase-1, another heme protein, was also regulated by oxygen level and Lon protease in the same mechanism as for CBS. Our findings provide a fundamental and general mechanism for oxygen-sensitive regulation of mitochondrial functions by linking oxygenation level to the accumulation/degradation of mitochondrial heme proteins.

Inactivation and Heme Degradation of Horseradish Peroxidase Induced by Discharge Plasma

AbstractThe effects of non‐thermal plasma at the argon gas–solution interface on horseradish peroxidase (HRP), a classical heme‐containing protein, were assessed quantitatively. Evidence from spectroscopic approaches showed that enzymatic activity together with the contents of heme and iron in HRP decreased dramatically and concomitantly upon plasma treatment. The plasma induced both long‐ and short‐lived reactive species which inactivated HRP in the solution. In particular, hydrogen peroxide broke the heme prosthetic group into fluorescent products, mainly dipyrrols and their derivatives, while other factors such as hydroxyl radicals and UV radiation generated during plasma discharge accelerated this heme protein degradation process.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl−) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV–visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10µM), precluding the enzyme from functioning at maximum activity (80–90% inhibition). MPO heme destruction occurred only in the presence of Cl−. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO–Compound I–Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe(V) redox intermediate of myeloperoxidase, to Compound II, which has Fe(IV) in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

P450cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450cin D241N mutant has been produced and found to be analogous to the P450cam D251N mutant. P450cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31±3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.

RECOMBINANT HORSERADISH PEROXIDASE FOR ANALYTICAL APPLICATIONS

86 Horseradish peroxidase isoenzyme C (HRP, EC 1.11.1.7), a hemeand Ca-contai ning glycoprotein, is a member of the superfa mily of plant peroxidases [1–3] that are able to utilize hydrogen peroxide to catalyze the one electron oxidation of a wide variety of organic and inorganic substrates. Whereas plant pe roxidases find interest for applications in va rious biotechnological processes, like bleaching and degradation of organic compounds (e.g. phenols and lignin), HRP specifically is used in analytical biochemistry and biotechnology as a marker enzyme for antibodies, DNA and low molecular mass analytes. Broad substrate specificity and high catalytic activity and stability determined the world-wide application of HRP in bioand immunosensors, chemiluminescent, fluorescent and electrochemical detection systems, DNA microarrays and biochips with HRP-based colori metric detection [4–6, 14, 56]. Progress made in peroxidase gene hetero logous expression opened up the prospect to study structure-function relationships by means of genetic engineering. The baculovirus expression system allowed production of recombinant HRP in soluble, active and glycosylated form [7], however, this system is laborious and not as wide spread as expression in E. coli. Expression of HRP in yeast indicated only very low expression of active enzyme [8]. Up to date for the production of wild-type as well as mutant recombinant HRP the E. coli expression system is mostly used [9, 10]. Recombinant HRP forms inclusion bodies containing only traces of heme if expressed in bacterial cytoplasm. Multistep refolding and reactivation of recombinant apo-peroxidase with the prosthetic heme group is complex as the protein contains four disulfide bonds and in addition must bind two Ca-ions per molecule. Moreover, the plant-derived enzyme contains 18% carbohydrates via 8 glycosylation sites. Crystal structure of recombinant HRP has been solved, revealing the presence of two domains formed by a total of ten α-helices [11]. Among of the factors leading to the formation of inclusion bodies upon expression of UDK 543.645.6:665.123

Redox activity distinguishes solid-state electron transport from solution-based electron transfer in a natural and artificial protein: cytochrome C and hemin-doped human serum albumin

Integrating proteins in molecular electronic devices requires control over their solid-state electronic transport behavior. Unlike "traditional" electron transfer (ET) measurements of proteins that involve liquid environments and a redox cycle, no redox cofactor is needed for solid-state electron transport (ETp) across the protein. Here we show the fundamental difference between these two approaches by macroscopic area measurements, which allow measuring ETp temperature dependence down to cryogenic temperatures, via cytochrome C (Cyt C), an ET protein with a heme (Fe-porphyrin) prosthetic group as a redox centre. We compare the ETp to electrochemical ET measurements, and do so also for the protein without the Fe (with metal-free porphyrin) and without porphyrin. As removing the porphyrin irreversibly alters the protein's conformation, we repeat these measurements with human serum albumin (HSA), 'doped' (by non-covalent binding) with a single hemin equivalent, i.e., these natural and artificial proteins share a common prosthetic group. ETp via Cyt C and HSA-hemin are very similar in terms of current magnitude and temperature dependence, which suggests similar ETp mechanisms via these two systems, thermally activated hopping (with ~0.1 eV activation energy) >190 K and tunneling by superexchange <190 K. Also, ET rates to and from the Fe redox centres (Fe(2+) <=> Fe(3+) + e(-)), measured by electrochemistry of HSA-hemin are only 4 times lower than those for Cyt C. However, while removing the Fe redox centre from the porphyrin ring markedly affects the ET rate, it hardly changes the ETp currents through these proteins, while removing the macrocycle (from HSA, which retains its conformation) significantly reduces ETp efficiency. These results show that solid-state ETp across proteins does not require the presence of a redox cofactor, and that while for ET the Fe ion is the main electron mediator, for ETp the porphyrin ring has this function.

Hemoproteins Designed from Scratch for O2 Transport, Co, No Signaling, Oxidoreductase and Cytochrome P450 Oxygenase Catalysis

We have designed and engineered a non-natural heme binding protein maquette with diverse heme ligations (bis-his, his-cys, single cys) and dramatically different spectroscopic and redox properties. Hemoproteins in nature perform diversified functions such as oxygen storage, oxygen transport, CO, NO signaling, gene regulation, drug metabolism. Hemoproteins achieve these diversified functions by modifying the redox properties of heme prosthetic group and by altered ligating amino acid to the central Fe metal or by use of different tetrapyrroles or by the nature of amino acids in the vicinity of heme. We have successfully designed a single chain tetra-helix bundle hemoproteins with bis-his, his-cys and single cys ligations, all display native absorption spectra of neuroglobin, CooA (transcription activator), other CO sensing enzymes and cytochrome P450 respectively. All these designed proteins form stable oxyferrous state with natural and synthetic iron porphyrins. These protein maquettes are readily expressable in E.coli. and holds great promise for doing P450 kind of reactions.

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis.

Direct electrochemistry of adsorbed proteins and bioelectrocatalysis at film electrode prepared from oppositely charged carbon nanoparticles

Carbon nanoparticulate film electrodes were prepared by alternative immersion of indium tin oxide plates into suspension of positively and negatively charged carbon nanoparticles. Their average thickness is in the range of 30–300nm as determined by atomic force microscopy and depends on the number of immersion and withdrawal steps. After adsorption of myoglobin the voltammetric signal corresponding to redox reaction of heme prosthetic group is observed. This electrode exhibits bioelectrocatalytic hydrogen peroxide reduction with cathodic current linearly dependent on concentration in millimolar range. After adsorption of glucose oxidase the electrode exhibits well defined voltammetric signal attributed to the electrochemical reaction of redox cofactor FAD. In oxygenated solution the cathodic current decreases and is linearly dependent on glucose concentration in the millimolar range. In the presence of mediator – ferrocene carboxylate, bioelectrocatalytic oxidation of glucose is seen. The magnitude of the redox voltammetric signal of both enzymes is roughly proportional to the amount of nanoparticulate material.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

PAS Domain Residues and Prosthetic Group Involved in BdlA-Dependent Dispersion Response by Pseudomonas aeruginosa Biofilms

Biofilm dispersion by Pseudomonas aeruginosa in response to environmental cues is dependent on the cytoplasmic BdlA protein harboring two sensory PAS domains and a chemoreceptor domain, TarH. The closest known and previously characterized BdlA homolog is the flavin adenine dinucleotide (FAD)-binding Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Here, we made use of alanine replacement mutagenesis of the BdlA PAS domain residues previously demonstrated to be essential for aerotaxis in Aer to determine whether BdlA is a potential sensory protein. Five substitutions (D14A, N23A, W60A, I109A, and W182A) resulted in a null phenotype for dispersion. One protein, the BdlA protein with the G31A mutation (BdlA-G31A), transmitted a constant signal-on bias as it rendered P. aeruginosa biofilms hyperdispersive. The hyperdispersive phenotype correlated with increased interaction of BdlA-G31A with the phosphodiesterase DipA under biofilm growth conditions, resulting in increased phosphodiesterase activity and reduced biofilm biomass accumulation. We furthermore demonstrate that BdlA is a heme-binding protein. None of the BdlA protein variants analyzed led to a loss of the heme prosthetic group. The N-terminal PASa domain was identified as the heme-binding domain of BdlA, with BdlA-dependent nutrient-induced dispersion requiring the PASa domain. The findings suggest that BdlA plays a role in intracellular sensing of dispersion-inducing conditions and together with DipA forms a regulatory network that modulates an intracellular cyclic d-GMP (c-di-GMP) pool to enable dispersion.