Probing structure-function relations in heme-containing oxygenases and peroxidases

Structural factors that influence functional properties are examined in the case of four heme enzymes: cytochrome P-450, chloroperoxidase, horseradish peroxidase, and secondary amine mono-oxygenase. The identity of the axial ligand, the nature of the heme environment, and the steric accessibility of the heme iron and heme edge combine to play major roles in determining the reactivity of each enzyme. The importance of synthetic porphyrin models in understanding the properties of the protein-free metal center is emphasized. The conclusions described herein have been derived from studies at the interface between biological and inorganic chemistry.     

Shunt bilirubin: evidence for two components

Studies with C(14)-labeled glycine and delta-aminolevulinic acid as heme-bilirubin precursors in man indicate that the early labeled or shunt bilirubin consists of two fractions. Fraction 1 requires 1 to 24 hours for maximum synthesis, is not dependent on marrow erythropoietic heme synthesis, and is possibly of anabolic origin (formed by a direct pathway from heme precursors). Fraction 2 requires 3 to 4 days for maximum production, is dependent on heme synthesis, and probably has its origin in the bone marrow, as a degradation product of red-cell heme.     

Heme and globin synthesis control: observations in vivo in beta thalassemia

After administration of glycine-2-(14)C to a patient with thalassemia, the specific activities of heme and globin of F hemoglobin were consistently higher than those of hemoglobin A. After reaching a maximum, the ratio of the specific activity of heme to that of globin remained constant within each hemoglobin. Explanations considered include dilution by preformed subunits, differential turnover of hemoglobins, and possibly more than one heme-synthesizing pool.     

捻转血矛线虫Hc-hrg-2拯救血红素缺陷型酵母生长表型

【目的】已有研究表明捻转血矛线虫（Haemonchus contortus）Hc-hrg-2属于血红素应答基因,高浓度血红素的刺激可导致该基因的转录水平上调,但其在血红素调控中的功能尚缺乏研究。研究通过同源重组技术构建血红素合成缺陷型酿酒酵母（Saccharomyces cerevisiae）hem1敲除株,通过异源表达Hc-hrg-2对该敲除株进行表型拯救试验,以期验证Hc-hrg-2参与细胞内血红素转运的功能。 【方法】以S. cerevisiae BY4741基因组为模板,设计引物,扩增获得hem1（SGD:S000002640）基因序列5' 和3' 侧翼区同源臂;同时以pYES2-CT质粒为模板设计引物,扩增获得筛选标记URA3序列;利用两次重叠PCR技术依次将测序正确的上游同源臂、URA3、下游同源臂序列串联成基因敲除组件,并通过醇沉法对敲除组件进行纯化。采用醋酸锂转化法将纯化的敲除组件转化至BY4741感受态细胞中, 经SD/- URA（含250 μmol·L^-1 5-氨基乙酰丙酸（ALA））培养基筛选,并利用多对引物进行PCR鉴定,以验证Δhem1敲除株的正确性。同时以含有H. contortus 浙江株Hc-hrg-2 序列（GenBank：MK371241）及其功能域缺失序列Hc-hrg-2(Δgst-n) 和Hc-hrg-2(Δgst-c) 的质粒为模板,利用特异性引物分别进行PCR扩增,通过无缝克隆将扩增得到的目的片段插入酵母pESC-LEU表达载体,经PCR鉴定以及测序验证后,采用醋酸锂转化法将正确的表达载体转化至Δhem1感受态细胞中,通过SD/- URA/- LEU（含250 μmol·L^-1 ALA）培养基筛选以及PCR鉴定验证阳性异源表达株的正确性。通过比较Δhem1敲除株及其各异源表达株在含有或不含有250 μmol·L^-1 ALA的SD/- URA/- LEU液体培养基中的生长情况,进一步验证敲除株的表型同时排除表达载体对表型的影响。用2%半乳糖对含有阳性表达质粒的敲除株进行诱导,取部分菌液进行超裂破碎,收集蛋白,通过Western Blot鉴定目的蛋白的表达;剩余菌体用去离子水重悬至OD₆₀₀ = 0.2,用去离子水进行5倍比稀释,取4 μL稀释后的菌液点至含有250 μmol·L^-1 ALA或者不同浓度血红素的诱导平板上,28℃培养2—3 d后比较敲除株的生长情况。【结果】成功获得hem1基因敲除株Δhem1,与野生株相比,Δhem1不能合成血红素,需外源添加ALA（250 μmol·L^-1）或血红素（≥ 10 μmol·L^-1）才能生长,且异源表达株和敲除株的表型一致。Western Blot结果表明2%半乳糖能诱导Hc-hrg-2及其功能域缺失基因在敲除株中成功表达,且表达Hc-hrg-2能够在低血红素浓度下（≤ 1 μmol·L^-1）促进酵母对血红素的摄取,拯救敲除株的生长缺陷,其硫氧还蛋白样结构域（GST-N）和谷胱甘肽S-转移酶C末端结构域（GST-C）的缺失会降低拯救的效果。【结论】捻转血矛线虫血红素应答基因Hc-hrg-2可促进细胞对血红素的摄取,其GST-N和GST-C功能域在该过程中发挥着重要作用,研究成果为后续深入研究捻转血矛线虫的血红素转运机制奠定基础。     

Cytochrome a3 structure in carbon monoxide-bound cytochrome oxidase

The iron-carbon monoxide stretching mode and the iron-carbon-oxygen bending mode in carbon monoxide-bound cytochrome oxidase have been assigned at 520 and 578 cm-1, respectively. The frequencies, widths, and intensities of these modes show that the Fe-C-O grouping in carbon monoxide-cytochrome a3 is linear but tilted from the normal to the heme plane; that the iron-histidine bond in both five- and six-coordinate cytochrome a3 is strained; and that the carbon monoxide and the proximal histidine each have characteristic, well-defined orientations in all molecules. These data can account for the binding affinities of carbon monoxide and dioxygen under physiological conditions.     

A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.     

Epinephrine reduction of heme: implication for understanding the transmission of an agonist stimulus

Alpha-adrenergic agonists that promote platelet aggregation were found to reduce ferric heme to ferrous heme. Agents that bind iron in heme inhibited epinephrine-induced platelet aggregation. It is proposed that epinephrine first binds to its receptor and then reduces an adjacent heme group to transmit its agonist stimulus.     

Iron-source preference of Staphylococcus aureus infections

Although bacteria use different iron compounds in vitGro, the possibility that microbes distinguish between these iron sources during infection has hitherto not been examined. We applied stable isotope labeling to detect source-specific iron by mass spectrometry and show that Staphylococcus aureus preferentially imports heme iron over transferrin iron. By combining this approach with computational genome analysis, we identified hts (heme transport system), a gene cluster that promotes preferred heme iron import by S. aureus. Heme iron scavenging by means of hts is required for staphylococcal pathogenesis in animal hosts, indicating that heme iron is the preferred iron source during the initiation of infection.     

Chemically induced porphyria: increased microsomal heme turnover after treatment with allylisopropylacetamide

Excessive induction of delta-aminolevulinic acid synthetase in rats after treatment with porphyria-inducing chemicals, such as allylisopropylacetamide, is accompanied by a decrease in microsomal heme and cytochrome P450 concentrations. Measurement of the radioactive decay after labeling of the heme moiety of submicrosomal particles shows increased breakdown of heme in rats treated with allylisopropylacetamide. The effects of allylisopropylacetamide on heme synthesis and heme turnover may be interrelated     

A steric mechanism for inhibition of CO binding to heme proteins

The crystal structures of myoglobin in the deoxy- and carbon monoxide-ligated states at a resolution of 1.15 angstroms show that carbon monoxide binding at ambient temperatures requires concerted motions of the heme, the iron, and helices E and F for relief of steric inhibition. These steps constitute the main mechanism by which heme proteins lower the affinity of the heme group for the toxic ligand carbon monoxide.     

Evidence for an inter-organismic heme biosynthetic pathway in symbiotic soybean root nodules

The successful symbiosis of soybean with Bradyrhizobium japonicum depends on their complex interactions, culminating in the development and maintenance of root nodules. A B. japonicum mutant defective in heme synthesis in culture was able to produce heme as a result of its symbiotic association with the soybean host. The bacterial mutant was incapable of synthesizing the committed heme precursor delta-aminolevulinic acid (ALA), but nodule plant cells formed ALA from glutamate. In addition, exogenous ALA was taken up by isolated nodule bacteria of the parent strain and of the mutant. It is proposed that bacterial heme found in nodules can be synthesized from plant ALA, hence segments of a single metabolic pathway are spatially separated into two organisms.     

Indoleacetic acid oxidase activity of apoperoxidase

The conventional activity of electrophoretically purified horseradish peroxidase toward guaiacol, pyrogallol, 2,6-dimethoxyphenol, and benzidine is abolished by removal of the heme prosthetic group with a mixture of cold acetone and hydrogen chloride. The apoenzyme, though devoid of peroxidase activity, retains its activity as an indoleacetic acid oxidase when it is supplied with 10(-5) mole of manganous ion and 2,4-dachlorophenol per liter. This oxidase activity is cyanide-sensitive; azide also inhibits under specific conditions of both pH and cofactor concentration. Partial restoration of the peroxidase activity by recombination of apoprotein with heme produces no effect on the oxidase activity, except that cofactors are no longer absolutely required. Therefore, it appears that the activity of peroxidase as an indoleacetic acid oxidase need not directly involve the heme prosthetic group, or that manganous ions and dichlorophenol can substitute for the heme group in the reaction between indoleacetic acid and oxidase.     

L-tryptophan: a common denominator of biochemical and neurological events of acute hepatic porphyria?

Hepatic porphyrias are disorders of heme synthesis characterized by genetically determined lesions of one of the key enzymes of heme synthesis. In carriers of such lesions, several factors (drugs, environmental chemicals, or diet) precipitate acute and often fatal attacks of neurologic dysfunction, which are promptly relieved by intravenous infusion of heme. However, the mechanism of such heme-induced amelioration remains elusive. To probe this mechanism, the biochemical events triggered by acute hepatic heme deficiency were examined in an animal model of chemically induced porphyria. Acute hepatic heme depletion in porphyric rats was found to impair hepatic tryptophan pyrrolase activity which, in turn, elevated tryptophan and 5-hydroxytryptamine turnover in the brain. These alterations in porphyric rats were dramatically reversed by parenteral heme administration. These findings suggest that increased tryptophan and 5-hydroxytryptamine in the nervous system may be responsible for the neurologic dysfunctions observed in humans with acute attacks of hepatic porphyria.     

ELECTROPHORETIC PATTERNS OF HEMOGLOBIN FROM FETAL MICE OF DIFFERENT INBRED STRAINS

Starch gel electrophoretic patterns of hemoglobins from fetal mice of seven inbred strains (two with single, five with diffuse adult hemoglobins), were compared with each other and with electrophoretic patterns of hemoglobins from adults of the same strains. The blood from 15-day-old fetuses of all strains contained four electrophoretically separable heme components. However, there seems to be a difference between strains with single and diffuse adult hemoglobin in the time of emergence of a clear adult pattern.     

Significance of mitochondria for porphyrin and heme biosynthesis

It is concluded that mitochondria are involved in three steps of porphyrin and heme biosynthesis-first, in the formation of delta-aminolevulinic acid from glycine and active succinate; second, in the synthesis of protoporphyrin; third, in the incorporation of iron into the porphyrin ring-that is, in heme formation.     

Fumarate reductase in the control of heme biosynthesis

A drug-induced stimulation of heme biosynthesis in mouse liver was accompanied by altered fumarate metabolism. In liver homogenate, fumarate 1,4-C(14) was incorporated, via succinate and succinyl coenzyme A, into heme at an accelerated rate. This pathway of fumarate utilization was inhibited by acetoacetate but not by beta-hydroxybutyrate. Fumarate reduction to succinate required reduced nicotinamide adenine dinucleotide. The enzyme fumarate reductase is suggested as a link between terminal oxidation and cellular control of the heme biosynthetic pathway.     

Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein

Erythropoietin (Epo), the hormone that stimulates red blood cell production, is synthesized in the kidney and liver in response to hypoxia. The human hepatoma cell line Hep3B regulates its production of Epo in a physiologic manner. Either hypoxia or cobalt chloride markedly increases expression of Epo mRNA as well as production of biologically active and immunologically distinct Epo protein. New protein synthesis is required before the induction of increased levels of hypoxia- or cobalt-induced Epo mRNA. Hypoxia, cobalt chloride, and nickel chloride appear to stimulate Epo production through a common pathway. The inhibition of Epo production at low partial pressures of oxygen by carbon monoxide provides evidence that a heme protein is integrally involved in the oxygen-sensing mechanism. This hypothesis is further supported by the finding that when heme synthesis is blocked, hypoxia-, cobalt-, and nickel-induced Epo production are all markedly inhibited. A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.     

Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity

Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes-epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis-work sequentially, and we propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade.