Abstract
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.
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Abbreviations
- AdoMet:
-
S-adenosyl-l-methionine
- δ-ALA:
-
δ-Aminolevulinic acid
- AMP:
-
Adenosine monophosphate
- BSA:
-
Bovine serum albumin
- CBS:
-
Cystathionine β-synthase
- Co:
-
Cobalt
- ETF:
-
Electron transfer flavoprotein
- ETF-QO:
-
ETF ubiquinone oxidoreductase
- FAD:
-
Flavin adenine dinucleotide
- Fe:
-
Iron
- GST:
-
Glutathione S-transferase
- ICP-OES:
-
Inductively coupled plasma optical emission spectroscopy.
- IPTG:
-
Isopropyl-β-d-1-thiogalactopyranoside
- IscS:
-
Cysteine desulfurase
- MIC:
-
Minimum inhibitory concentration
- PLP:
-
Pyridoxal-5′-phosphate
- PPIX:
-
Protoporphyrin IX
- SEM:
-
Standard error of the mean
- ThiH:
-
Tyrosine lyase
- WT:
-
Wild type
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Acknowledgements
This work was supported by Postdoctoral Fellowship 0920079G from the American Heart Association (to TM), by National Institutes of Health Grant HL065217 (to JPK), by American Heart Association Grant In-Aid 09GRNT2110159 and by a grant from the Jerome Lejeune Foundation (to JPK). The authors thank Dr. James P. McEvoy (Regis University, Denver) for assistance with measuring of cells oxygen uptake, Cheryl K. Peck (University of Colorado, Denver) for assistance with GC/MS analysis and Katherine M. Freeman and Judith N. Burstyn (University of Wisconsin, Madison) for helpful comments and critical reading of the manuscript.
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Majtan, T., Frerman, F.E. & Kraus, J.P. Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation. Biometals 24, 335–347 (2011). https://doi.org/10.1007/s10534-010-9400-7
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DOI: https://doi.org/10.1007/s10534-010-9400-7