caa a22 4500 445322128 CHVBK 20180317142707.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s10534-010-9400-7 doi (NATIONALLICENCE)springer-10.1007/s10534-010-9400-7 Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation [Elektronische Daten] [Tomas Majtan, Frank Frerman, Jan Kraus] Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe-S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS. Springer Science+Business Media, LLC., 2010 Cobalt toxicity nationallicence Cobalt protoporphyrin IX nationallicence Protein expression nationallicence Heme replacement nationallicence Chelatase nationallicence Fe-S cluster nationallicence Cystathionine beta-synthase nationallicence Respiration nationallicence AdoMet : S-adenosyl-l-methionine nationallicence δ-ALA : δ-Aminolevulinic acid nationallicence AMP : Adenosine monophosphate nationallicence BSA : Bovine serum albumin nationallicence CBS : Cystathionine β-synthase nationallicence Co : Cobalt nationallicence ETF : Electron transfer flavoprotein nationallicence ETF-QO : ETF ubiquinone oxidoreductase nationallicence FAD : Flavin adenine dinucleotide nationallicence Fe : Iron nationallicence GST : Glutathione S-transferase nationallicence ICP-OES : Inductively coupled plasma optical emission spectroscopy nationallicence IPTG : Isopropyl-β-d-1-thiogalactopyranoside nationallicence IscS : Cysteine desulfurase nationallicence MIC : Minimum inhibitory concentration nationallicence PLP : Pyridoxal-5′-phosphate nationallicence PPIX : Protoporphyrin IX nationallicence SEM : Standard error of the mean nationallicence ThiH : Tyrosine lyase nationallicence WT : Wild type nationallicence Majtan Tomas Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut Frerman Frank Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut Kraus Jan Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut BioMetals Springer Netherlands 24/2(2011-04-01), 335-347 0966-0844 24:2<335 2011 24 10534 https://doi.org/10.1007/s10534-010-9400-7 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10534-010-9400-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Majtan Tomas Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut NATIONALLICENCE 700 1- Frerman Frank Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut NATIONALLICENCE 700 1- Kraus Jan Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave, 80045, Aurora, CO, USA aut NATIONALLICENCE 773 0- BioMetals Springer Netherlands 24/2(2011-04-01), 335-347 0966-0844 24:2<335 2011 24 10534 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer