Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Zinc is a constituent of bovine heart cytochrome c oxidase preparations

Cytochrome c oxidase preparations from bovine heart muscle contain 1 zinc per 2 irons. Metal contents of nine preparations determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) show that Cu, Fe and Zn are the only metals present in significant amounts with average Cu/Fe, Fe/Zn, and Cu/Zn atom ratios of 1.3, 2.1 and 2.8, respectively. Removal of adventitious copper results in a Cu:Fe:Zn stoichiometry of 2:2:1. The zinc is tightly bound. Dialysis against a solution of 1,10-phenanthroline at pH 7.4 or an acidic buffer (pH 4.4) does not remove Zn. Dialysis against 0.8 M KCN at pH 10 causes partial loss of both Cu and Zn. This is the first evidence for the presence of Zn in a cytochrome c oxidase.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mössbauer spectra of the heme peptide (HP) 1–50 and the heme peptide:non-heme peptide (NHP) non-covalent complex 1–50:51–104 derived from cytochrome c: evidence for cytochrome c iron site solvation in aqueous solution

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.     

Fractionation of erytroblasts with affinitymediated modifications of their electrical properties using counter-current distribution

Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.Abbreviations BVR biliverdin reductase - DMEM Dulbecco's modified Eagles medium - DMSO dimethyl sulfoxide - HO heme oxygenase - 1xSSC a solution of 0.015 M sodium citrate/0.15 sodium chloride     

鸭疫里氏杆菌hemH基因功能初步鉴定及其缺失株的转录组学分析

血红素是绝大多数细菌生长繁殖所必需的一种营养物质,其参与了细菌多种重要的生理过程。细菌可以通过自身合成和从外界摄取2种方式获得血红素。然而过多的血红素对细菌是有毒性的,细菌则会利用外排、螯合和降解等多种方式减轻血红素毒性。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染禽类的革兰氏阴性菌,前期研究表明该菌可通过转运的方式从外界环境摄取血红素。然而,是否该菌也可以自身合成血红素未知。基因组分析发现鸭疫里氏杆菌ATCC11845菌株中的基因RA0C＿RS08070编码铁螯合酶(ferrochelatase) HemH,是参与将铁插入卟啉中心,形成血红素的关键酶。hem H缺失后会导致铁离子和卟啉的积累,对细菌造成毒性。【目的】为鉴定Hem H在合成血红素中的功能及查找参与鸭疫里氏杆菌铁离子和卟啉解毒相关基因。【方法】本研究构建了RAATCC11845Δhem H缺失株,并检测亲本株和hem H缺失株在GCB以及GCB添加血红蛋白液体培养基中的生长情况;随后对亲本株和hem H缺失株进行转录组测序并进行比较分析。【结果】RAATCC11845Δhem H缺失...     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.