Identification of the haem-binding subunit of cytochrome b-245.
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ABSTRACT:
SUBMITTER: Nugent JH
PROVIDER: S-EPMC1133673 | biostudies-other | 1989 Dec
REPOSITORIES: biostudies-other
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Project description:Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (NrfI, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as CX(2)CH (by CcsA2), CX(2)CK (by NrfI) and CX(15)CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of NrfI and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs.
| S-EPMC3068706 | biostudies-literature
Project description:Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX(2)CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active-site HBMs (CX(2)CK or CX(2)CH). W. succinogenes CcsA2 was found to attach haem to standard CX(2)CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX(2)CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX(15)CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes.
| S-EPMC3414424 | biostudies-literature
Project description:Cyanide is a well known potent inhibitor of haem proteins, including haem oxygenase (HO). Generally, cyanide coordinates to the ferric haem iron with a linear binding geometry; the Fe-C-N angle ranges from 160 to 180 degrees . The Fe-C-N angle observed in the crystal structure of haem-HO bound to cyanide prepared at alkaline pH was 166 degrees . Here, it is reported that cyanide can bind to the haem iron in HO in a bent mode when the ternary complex is prepared at neutral pH; a crystal structure showed that the Fe-C-N angle was bent by 47 degrees . Unlike the ternary complex prepared at alkaline pH, in which the haem group, including the proximal ligand and the distal helix, was displaced upon cyanide binding, the positions of the haem group and the distal helix in the complex prepared at neutral pH were nearly identical to those in haem-HO. Cyanide that was bound to haem-HO with a bent geometry was readily photodissociated, whereas that bound with a linear geometry was not photodissociated. Thus, alternative cyanide-binding modes with linear and bent geometries exist in the crystalline state of haem-HO.
| S-EPMC2335079 | biostudies-literature
Project description:BackgroundX-linked chronic granulomatous disease (X-CGD) is an immunodeficiency disorder caused by defects in the gp91phox subunit that leads to life-threatening infections. We aimed to identify CYBB gene mutations and study clinical phenotypes in Iranian patients with probable X-CGD.MethodsWe studied four unrelated Iranian patients with probable X-CGD and their families recruited in several years. We isolated genomic DNA from whole blood and performed Sanger sequencing in the CYBB gene's coding and flanking regions. We also performed pathogenicity predictions using in silico tools.ResultsWe detected four different mutations, including a novel insertion mutation in exon 5 (p.Ile117AsnfsX6), in the patient. Bioinformatics analysis confirmed the pathogenic effect of this mutation. We predicted protein modeling and demonstrated lost functional domains. The patient with the insertion mutation presented pneumonia and acute sinusitis during his life. We also detected three other known nonsense mutations (p.Arg157Ter, p.Arg226Ter, and p.Trp424Ter) in the CYBB gene. The patient with p.Arg157Ter developed lymphadenitis and pneumonia. Moreover, the patient with inflammatory bowel disease showed p.Arg226Ter and the patient with tuberculosis presented p.Trp424Ter. We detected different clinical features in the patients compared to other Iranian patients with the same mutations.ConclusionOur results expand the genetic database of patients with X-CGD from Iran and make it much easier and faster to identify patients with X-CGD. Our results also help to detect carriers and enable prenatal diagnosis in high-risk families as a cost-effective strategy.
| S-EPMC7891530 | biostudies-literature
Project description:The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.
| S-EPMC1153509 | biostudies-other
Project description:Alkyl isonitriles, R-NC, have previously been shown to ligate the heme (haem) iron of cytochromes P450 in both accessible oxidation states (ferrous, Fe2+, and ferric, Fe3+). Herein, the preparation of four steroid-derived isonitriles and their interactions with several P450s, including the steroidogenic CYP17A1 and CYP106A2, as well as the more promiscuous drug metabolizers CYP3A4 and CYP2D6, is described. It was found that successful ligation of the heme iron by the isonitrile functionality for a given P450 depends on both the position and stereochemistry of the isonitrile on the steroid skeleton. Spectral studies indicate that isonitrile ligation of the ferric heme is stable upon reduction to the ferrous form, with reoxidation resulting in the original complex. A crystallographic structure of CYP17A1 with an isonitrile derived from pregnanalone further confirmed the interaction and identified the absolute stereochemistry of the bound species.
| S-EPMC10475101 | biostudies-literature
Project description:BackgroundThe association between short stature, undernutrition and the risk to cardiovascular disease has been clinically established. Genetic factor, particularly the variants in cytochrome b-245 alpha chain (CYBA) gene, which alter the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase level, might affect arterial function. This study aimed to observe the association between single nucleotide variants (SNVs) of the CYBA gene and the arterial function of short stature children to understand the reason why some people with short stature develop cardiovascular disease.MethodsA total of 142 genomic deoxyribonucleic acid (DNA) samples have been collected from short stature children in Brebes, Central Java, Indonesia. Four common single-nucleotide polymorphisms (SNPs): C242T (rs4673), A640G (rs1049255), -930A>G (rs9932581) and *49A>G (rs7195830) in the CYBA gene were examined using TaqMan allelic discrimination assay. The arterial function was measured using transthoracic echocardiography and described as aortic stiffness and distensibility index. Statistical analysis was done to find a significant difference in arterial function between genotypes of each SNV.ResultsA P-value of < 0.05 was considered significant. In rs9932581 (-930A>G) of CYBA gene, the subjects with GG genotype were found to have significantly lower arterial stiffness and higher distensibility compared to AA and AG genotypes. No significant difference was found in the other SNVs.ConclusionThe GG genotype in rs9932581 of the CYBA gene might have a protective effect on cardiovascular disease in short stature children.
| S-EPMC8683098 | biostudies-literature
Project description:Cytochrome b-245 is a glycoprotein. It runs as a broad band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its apparent Mr varies with the concentration of acrylamide. It stained positively with Schiff reagent and with silver stains after oxidation with periodic acid. It preferentially bound the lectin of Phaseolus vulgaris (type III), and cleavage of carbohydrate with endoglycosidase F resulted in a sharp band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 55000 G.l.c. analysis of carbohydrate showed this to account for about 15% of the Mr and N-acetylglucosamine and galactose to be the major sugars.
| S-EPMC1144906 | biostudies-other
Project description:Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.
| S-EPMC1147335 | biostudies-other
Project description:Characterization of a novel haem-binding protein that is secreted by two Haemophilus haemolyticus isolates, BW1 and RHH122, and that inhibits the growth of the pathogen non-typeable Haemophilus influenzae (NTHi) in vitro.
2019-05-29 | PXD013687 | Pride