CO2/HCO3- Accelerates Iron Reduction through Phenolic Compounds

Abstract

Iron is a vital mineral for almost all living organisms and has a pivotal role in central metabolism. Despite its great abundance on earth, the accessibility for microorganisms is often limited, because poorly soluble ferric iron (Fe³⁺) is the predominant oxidation state in an aerobic environment. Hence, the reduction of Fe³⁺ is of essential importance to meet the cellular demand of ferrous iron (Fe²⁺) but might become detrimental as excessive amounts of intracellular Fe²⁺ tend to undergo the cytotoxic Fenton reaction in the presence of hydrogen peroxide. We demonstrate that the complex formation rate of Fe³⁺ and phenolic compounds like protocatechuic acid was increased by 46% in the presence of HCO₃^- and thus accelerated the subsequent redox reaction, yielding reduced Fe²⁺ Consequently, elevated CO₂/HCO₃^- levels increased the intracellular Fe²⁺ availability, which resulted in at least 50% higher biomass-specific fluorescence of a DtxR-based Corynebacterium glutamicum reporter strain, and stimulated growth. Since the increased Fe²⁺ availability was attributed to the interaction of HCO₃^- and chemical iron reduction, the abiotic effect postulated in this study is of general relevance in geochemical and biological environments.IMPORTANCE In an oxygenic environment, poorly soluble Fe³⁺ must be reduced to meet the cellular Fe²⁺ demand. This study demonstrates that elevated CO₂/HCO₃^- levels accelerate chemical Fe³⁺ reduction through phenolic compounds, thus increasing intracellular Fe²⁺ availability. A number of biological environments are characterized by the presence of phenolic compounds and elevated HCO₃^- levels and include soil habitats and the human body. Fe²⁺ availability is of particular interest in the latter, as it controls the infectiousness of pathogens. Since the effect postulated here is abiotic, it generally affects the Fe²⁺ distribution in nature.

Keywords: Corynebacterium glutamicum; DtxR; bicarbonate; carbon dioxide; iron homeostasis; iron reduction; pathogens.

FIG 1

Biomass formation of C. glutamicum FEM3 (A) and biomass-specific fluorescence after 24 h of cultivation in a parallel bioreactor setup (B). C. glutamicum FEM3 was cultivated aerobically in minimal medium with 20 g glucose liter⁻¹ with synthetic air containing 20% CO₂ (21% O₂, 59% N₂) or ambient air (0.04% CO₂). Data points (A) and bars (B) represent mean values, and error bars indicate standard deviations for three biological independent replicates.

FIG 2

(A) Shaking flask cultivations of wild-type (WT) C. glutamicum in minimal medium with 20 g glucose liter⁻¹ without supplement (reference), with 195 μM PCA, 50 mM NaHCO₃ or a combination of both supplements. (B) Biomass-specific fluorescence of C. glutamicum FEM3 after 24 h of cultivation with the indicated supplements. Data points and bars represent mean values with error bars indicating standard deviations for 3 to 10 independent biological replicates. Values that are significantly different by a two-sample t test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 3

(A) Kinetic analysis of the Fe²⁺-BPS complex formation at 534 nm with 19.5 μM PCA and/or 50 mM HCO₃⁻. (B) Relative PCA degradation over time in the presence and absence of 50 mM NaHCO₃. Data points and bars represent mean values with error bars indicating standard deviations for three to six independent replicates.

FIG 4

Kinetic analysis of the Fe²⁺-BPS complex formation at 534 nm with different functionalized aromatic compounds. Data points represent mean values with error bars indicating standard deviations of three to six independent replicates.

FIG 5

Complex formation between Fe³⁺ and PCA in the presence and absence of 50 mM NaHCO₃. (A) The kinetics of complex formation was monitored by an increase of the absorbance at 560 nm. (B) Wavelength of the maximum absorbance (λ_max) of the Fe³⁺-PCA complexes formed in the presence and absence of NaHCO₃ correlated with pH. Data points represent mean values, and error bars indicate standard deviations for three to five independent replicates.