BioMetals

, Volume 24, Issue 2, pp 335–347

Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation

Article

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.

Keywords

Cobalt toxicity Cobalt protoporphyrin IX Protein expression Heme replacement Chelatase Fe–S cluster Cystathionine beta-synthase Respiration 

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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Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Tomas Majtan
    • 1
    • 2
  • Frank E. Frerman
    • 1
  • Jan P. Kraus
    • 1
  1. 1.Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC)University of Colorado at DenverAuroraUSA
  2. 2.Department of Genomics and Biotechnology, Institute of Molecular BiologySlovak Academy of SciencesBratislavaSlovakia

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