Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme.

Heme has a variety of functions, from electronic reactions to binding gases, which makes it useful in medical treatments, dietary supplements, and food processing. In recent years, whole-cell system-based heme biosynthesis methods have been continuously explored and optimized as an alternative to the low-yield, lasting, and adverse ecological environment of chemical synthesis methods. This method relies on two biosynthetic pathways of microbial precursor 5-aminolevulinic acid (C4, C5) and three known downstream biosynthetic pathways of heme. This paper reviews the genetic and metabolic engineering strategies for heme production in recent years by optimizing culture conditions and techniques from different microorganisms. Specifically, we summarized and analyzed the possibility of using biosensors to explore new strategies for the biosynthesis of heme from the perspective of synthetic biology, providing a new direction for future exploration.

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins.

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme-albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins.

Spontaneous reduction of iron(III)porphyrin to iron(II)porphyrin-CO complex in mouse circulation.

Iron(II/III)porphyrin/cyclodextrin inclusion complexes serve as hemoprotein models in vivo. Here we showed the iron(III)porphyrin complex to be spontaneously reduced to its iron(II) state in mouse circulation. The reduced complex bound endogenous CO from carboxyhemoglobin, which was followed by urinary excretion. The natural reduction system was found to be effective for synthetic heme-model compounds.

Improved biosynthesis of heme in Bacillus subtilis through metabolic engineering assisted fed-batch fermentation.

BACKGROUND: Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. RESULTS: The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. CONCLUSIONS: Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

A Spin-Dependent Model for Multi-Heme Bacterial Nanowires.

The electrical properties of conductive heme-based nanowires found in Geobacter sulfurreducens bacteria were investigated using spin-dependent density functional theory (DFT). Molecular orbitals were generated using a restricted open-shell model which was solved by applying constraints to the spin-separated unrestricted open-shell model. Charge transport was simulated at different length scales ranging from individual heme sites up to the monomer unit of the nanowire, looking at hopping and tunneling between neighboring heme porphyrins with different Fe oxidation states. The resulting spin-dependent DFT results indicate that tunneling rates between heme sites are highly dependent on oxidation state and transport pathway modeled. The model demonstrates the importance of spin dependence for electron hopping, oxidation state, and decoherence transport in cytochromes. Applying non-equilibrium's Green's function to the system confirmed a substantial decrease in decoherent charge transport for the oxidized molecule at lower Fermi energies. In addition, partial or full oxidation of the heme sites in the nanowire created conditions for spin-dependent transport that can be exploited for spin-filtering effects in nanodevices.

Reduced water-holding capacity of beef during refrigeration is associated within creased heme oxygenase 1 expression, oxidative stress and ferroptosis.

Low molecular weight iron (LMW-Fe)-mediated oxidative stress from heme degradation may reduce beef water-holding capacity (WHC). However, the underlying mechanism of heme degradation is still unknown. In the present study, we assessed the WHC, tissue morphology, reactive oxygen species (ROS), apoptosis, heme oxygenase(HMOX) 1 expression, and ferroptosis characteristics of beef chilled at 4 °C for 6 days. Results showed that water loss increased and WHC decreased during beef storage (P < 0.05). Increased protein and mRNA expression of HMOX1 promoted the decomposition of heme and facilitated the liberation of iron ions (P < 0.05), and excess LMW-Fe was associated with ROS formation, depletion of glutathione, and inhibition of glutathione peroxidase 4 activity (P < 0.05). Muscle tissue showed typical features of ferroptosis, including expression of ferroptosis-related genes, malondialdehyde accumulation, and structural damage to mitochondria (P < 0.05). It was also found that HMOX1 and the heme pathway-mediated ferroptosis were associated with structural changes in myofibrils and reduced WHC in chilled beef.

Carbon Monoxide-Sensing Transcription Factors: Regulators of Microbial Carbon Monoxide Oxidation Pathway Gene Expression.

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate. In this review, we examine two known heme-dependent transcription factors, CooA and RcoM, that regulate inducible CO metabolism pathways in anaerobic and aerobic microorganisms. We provide an analysis of the known physiological and genomic contexts of these sensors and employ this analysis to contextualize known biochemical properties. In addition, we describe a growing list of putative transcription factors associated with CO metabolism that potentially use cofactors other than heme to sense CO.

5-ALA treatment increases intracellular heme levels and enhances CYP3A4 activity in genome-edited Caco-2 cells.

In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4.

Discovery of novel sulfonamide chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors.

A series of chromone-oxime derivatives containing piperazine sulfonamide moieties were designed, synthesized and evaluated for their inhibitory activities against IDO1. These compounds displayed moderate to good inhibitory activity against IDO1 with IC50 values in low micromolar range. Among them, compound 10m bound effectively to IDO1 with good inhibitory activities (hIDO1 IC50 = 0.64 µM, HeLa IDO1 IC50 = 1.04 µM) and were selected for further investigation. Surface plasmon resonance analysis confirmed the direct interaction between compound 10m and IDO1 protein. Molecular docking study of the most active compound 10m revealed key interactions between 10m and IDO1 in which the chromone-oxime moiety coordinated to the heme iron and formed several hydrogen bonds with the porphyrin ring of heme and ALA264, consistent with the observation by UV-visible spectra that 10m induced a Soret peak shift from 403 to 421 nm. Moreover, compound 10m exhibited no cytotoxicity at its effective concentration in MTT assay. Consistently, in vivo assays results demonstrated that 10m displayed potent antitumor activity with low toxicity in CT26 tumor-bearing Balb/c mice, in comparison with 1-methyl-l-tryptophan (1-MT) and 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L). In brief, the results suggested that chromone-oxime derivatives containing sulfonamide moieties might serve as IDO1 inhibitors for the development of new antitumor agents.

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins.

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.

Mechano-control of Extracellular Electron Transport Rate via Modification of Inter-heme Coupling in Bacterial Surface Cytochrome.

Bacterial outer-membrane multi-heme cytochromes (OMCs) mediate extracellular electron transport (EET). While heme alignment dictates the rate of EET, control of inter-heme coupling in a single OMC remains challenging, especially in intact cells. Given that OMCs diffuse and collide without aggregation on the cell surface, the overexpression of OMCs could increase such mechanical stress to impact the OMCs' protein structure. Here, the heme coupling is modified via mechanical interactions among OMCs by controlling their concentrations. Employment of whole-cell circular dichroism (CD) spectra of genetically engineered Escherichia coli reveals that the OMC concentration significantly impacts the molar CD and redox property of OMCs, resulting in a 4-fold change of microbial current production. The overexpression of OMCs increased the conductive current across the biofilm on an interdigitated electrode, indicating that a higher concentration of OMCs causes more lateral inter-protein electron hopping via collision on the cell surface. The present study would open a novel strategy to increase microbial current production by mechanically enhancing the inter-heme coupling.

Interactions of azole-based inhibitors with human heme oxygenase.

Human heme oxygenase-1 (hHO-1) plays a crucial role in human physiology because of its ability to metabolize free heme. The heme degradation products, biliverdin and bilirubin, were shown to have protective antioxidant properties in cells. In the context of cancer, hHO-1 function grants cancer cells defense from standard chemotherapy treatments, leading to the development of azole-based inhibitors that target hHO-1 for potential anticancer therapy. This work reports experimental and theoretical characterization of interactions between three azole-based inhibitors and the active site of hHO-1. It was found that all three compounds have Kd values within the µM order. The electronic absorption and resonance Raman (rR) spectra indicated that they bind to the ferric heme and coordinate through a nitrogen atom. rR measurements revealed varying effects of inhibitors on the geometry of heme vinyl groups in the ferric form of hHO-1. Changes in peripheral group orientation are known to affect heme redox potential, and consequently can reflect the inhibitory properties of studied azoles. The subsequent docking studies showed that inhibitors with lower Kd values are located close to two vinyl groups, while the compound with higher Kd is situated near only one, consistent with the rR studies. Finally, the rR studies of the CO adducts showed that the inhibitors bind to the heme in a reversible manner. Altogether, the combination of ligand binding studies, UV-Vis and rR spectroscopies, as well as computational approach revealed an importance of the steric hindrance imposed by the inhibitor's side chain.

Wolbachia Ferrochelatase as a potential drug target against filarial infections.

Filarial infections are among the world's most disturbing diseases caused by 3 major parasitic worms; Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi, affecting more than 500 million people worldwide. Currently used drugs for mass drug administration (MDA) have been met with several challenges including the development of complications in individuals with filaria co-infections and parasitic drug resistance. The filarial endosymbiont, Wolbachia, has emerged as an attractive therapeutic target for filariasis elimination, due to the dependence of the filaria on this endosymbiont for survival. Here, we target an important enzyme in the Wolbachia heme biosynthetic pathway (ferrochelatase), using high-throughput virtual screening and molecular dynamics with MM-PBSA calculations. We identified four drug candidates; Nilotinib, Ledipasvir, 3-benzhydryloxy-8-methyl-8-azabicyclo[3.2.1]octane, and 2-(4-Amino-piperidin-1-yl)-ethanol as potential small molecules inhibitors as they could compete with the enzyme's natural substrate (Protoporphyrin IX) for active pocket binding. This prevents the worm from receiving the heme molecule from Wolbachia for their growth and survival, resulting in their death. This study which involved targeting enzymes in biosynthetic pathways of the parasitic worms' endosymbiont (Wolbachia), has proven to be an alternative therapeutic option leading to the discovery of new drugs, which will help facilitate the elimination of parasitic infections.

Meconium-stained amniotic fluid.

Green-stained amniotic fluid, often referred to as meconium-stained amniotic fluid, is present in 5% to 20% of patients in labor and is considered an obstetric hazard. The condition has been attributed to the passage of fetal colonic content (meconium), intraamniotic bleeding with the presence of heme catabolic products, or both. The frequency of green-stained amniotic fluid increases as a function of gestational age, reaching approximately 27% in post-term gestation. Green-stained amniotic fluid during labor has been associated with fetal acidemia (umbilical artery pH <7.00), neonatal respiratory distress, and seizures as well as cerebral palsy. Hypoxia is widely considered a mechanism responsible for fetal defecation and meconium-stained amniotic fluid; however, most fetuses with meconium-stained amniotic fluid do not have fetal acidemia. Intraamniotic infection/inflammation has emerged as an important factor in meconium-stained amniotic fluid in term and preterm gestations, as patients with these conditions have a higher rate of clinical chorioamnionitis and neonatal sepsis. The precise mechanisms linking intraamniotic inflammation to green-stained amniotic fluid have not been determined, but the effects of oxidative stress in heme catabolism have been implicated. Two randomized clinical trials suggest that antibiotic administration decreases the rate of clinical chorioamnionitis in patients with meconium-stained amniotic fluid. A serious complication of meconium-stained amniotic fluid is meconium aspiration syndrome. This condition develops in 5% of cases presenting with meconium-stained amniotic fluid and is a severe complication typical of term newborns. Meconium aspiration syndrome is attributed to the mechanical and chemical effects of aspirated meconium coupled with local and systemic fetal inflammation. Routine naso/oropharyngeal suctioning and tracheal intubation in cases of meconium-stained amniotic fluid have not been shown to be beneficial and are no longer recommended in obstetrical practice. A systematic review of randomized controlled trials suggested that amnioinfusion may decrease the rate of meconium aspiration syndrome. Histologic examination of the fetal membranes for meconium has been invoked in medical legal litigation to time the occurrence of fetal injury. However, inferences have been largely based on the results of in vitro experiments, and extrapolation of such findings to the clinical setting warrants caution. Fetal defecation throughout gestation appears to be a physiologic phenomenon based on ultrasound as well as in observations in animals.

Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition in Staphylococcus aureus.

Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron limitation, the invasive pathogen Staphylococcus aureus uses the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane of S. aureus possesses special compartments (functional membrane microdomains [FMMs]) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs.

Multiple C-C Bond Cleavage Reactions Catalyzed by Tolyporphin Tetrapyrrole Biosynthetic Enzymes.

Tolyporphin A is an unusual tetrapyrrole secondary metabolite containing pendant deoxysugars and unsubstituted pyrrole ß sites. Herein, we describe the biosynthesis of the tolyporphin aglycon core. HemF1 catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III, an intermediate in heme biosynthesis. HemF2 then processes the two remaining propionate groups to generate a tetravinyl intermediate. All four vinyl groups from the macrocycle are truncated by TolI via repeated C-C bond cleavages to generate the unsubstituted pyrrole ß sites of tolyporphins. This study illustrates how the unprecedented C-C bond cleavage reactions branch from canonical heme biosynthesis to produce tolyporphins.

Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily.

Nonheme diiron enzymes harness the chemical potential of oxygen to catalyze challenging reactions in biology. In their resting state, these enzymes have a diferrous cofactor that is coordinated by histidine and carboxylate ligands. Upon exposure to oxygen, the cofactor oxidizes to its diferric state forming a peroxo- adduct, capable of catalyzing a wide range of oxidative chemistries such as desaturation and heteroatom oxidation. Despite their versatility and prowess, an emerging subset of nonheme diiron enzymes has inherent cofactor instability making them resistant to structural characterization. This feature is widespread among members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily. HDOs have a flexible core structure that remodels upon metal binding. Although ~9600 HDOs have been unearthed, few have undergone functional characterization to date. In this chapter, we describe the methods that have been used to characterize the HDO N-oxygenase, SznF. We demonstrate the overexpression and purification of apo-SznF and methodology specifically designed to aid in obtaining an X-ray structure of holo-SznF. We also describe the characterization of the transient SznF-peroxo-Fe(III)2 complex by stopped-flow absorption and Mössbauer spectroscopies. These studies provide the framework for the characterization of new members of the HDO superfamily.

Microbial Synthesis of Heme b: Biosynthetic Pathways, Current Strategies, Detection, and Future Prospects.

Heme b, which is characterized by a ferrous ion and a porphyrin macrocycle, acts as a prosthetic group for many enzymes and contributes to various physiological processes. Consequently, it has wide applications in medicine, food, chemical production, and other burgeoning fields. Due to the shortcomings of chemical syntheses and bio-extraction techniques, alternative biotechnological methods have drawn increasing attention. In this review, we provide the first systematic summary of the progress in the microbial synthesis of heme b. Three different pathways are described in detail, and the metabolic engineering strategies for the biosynthesis of heme b via the protoporphyrin-dependent and coproporphyrin-dependent pathways are highlighted. The UV spectrophotometric detection of heme b is gradually being replaced by newly developed detection methods, such as HPLC and biosensors, and for the first time, this review summarizes the methods used in recent years. Finally, we discuss the future prospects, with an emphasis on the potential strategies for improving the biosynthesis of heme b and understanding the regulatory mechanisms for building efficient microbial cell factories.

Biocatalysis for biorefineries: The case of dye-decolorizing peroxidases.

Dye-decolorizing Peroxidases (DyPs) are heme-containing enzymes in fungi and bacteria that catalyze the reduction of hydrogen peroxide to water with concomitant oxidation of various substrates, including anthraquinone dyes, lignin-related phenolic and non-phenolic compounds, and metal ions. Investigation of DyPs has shed new light on peroxidases, one of the most extensively studied families of oxidoreductases; still, details of their microbial physiological role and catalytic mechanisms remain to be fully disclosed. They display a distinctive ferredoxin-like fold encompassing anti-parallel ß-sheets and α-helices, and long conserved loops surround the heme pocket with a role in catalysis and stability. A tunnel routes H2O2 to the heme pocket, whereas binding sites for the reducing substrates are in cavities near the heme or close to distal aromatic residues at the surface. Variations in reactions, the role of catalytic residues, and mechanisms were observed among different classes of DyP. They were hypothetically related to the presence or absence of distal H2O molecules in the heme pocket. The engineering of DyPs for improved properties directed their biotechnological applications, primarily centered on treating textile effluents and degradation of other hazardous pollutants, to fields such as biosensors and valorization of lignin, the most abundant renewable aromatic polymer. In this review, we track recent research contributions that furthered our understanding of the activity, stability, and structural properties of DyPs and their biotechnological applications. Overall, the study of DyP-type peroxidases has significant implications for environmental sustainability and the development of new bio-based products and materials with improved end-of-life options via biodegradation and chemical recyclability, fostering the transition to a sustainable bio-based industry in the circular economy realm.

A Biochemical Deconstruction-Based Strategy to Assist the Characterization of Bacterial Electric Conductive Filaments.

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.