[27] Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples
Publisher Summary
This chapter discusses the rapid colorimetric micro-method for the quantitation of complexed iron in biological samples. The array of methods available for quantitating iron in an experimental sample often bewilders the investigator occasionally faced with the need for such quantitation. However, because of the development of more sensitive chromophoric chelators and simpler techniques, new methodologies continually evolve. This chapter describes one of these more recent methodologies for the quantitative determination of iron with ease, sensitivity, and simplicity. Methods commonly employed for the quantitation of complexed iron in biological samples rely on an initial treatment, which releases the complexed iron for its subsequent quantitative determination. Dry ashing in a furnace or wet ashing with hot concentrated acid releases the iron, but both procedures take time and present hazards, particularly for the researcher with only an occasional need to determine the iron content of a biological sample. This chapter explains that the simplicity of the assay procedure easily accommodates a preliminary investigation to examine for possible interference or contamination by any buffer system under consideration. In conclusion, the procedure detailed in this chapter offers the investigator without access to more sophisticated instrumentation, a simple and sensitive colorimetric method for the quantitation of iron in biological material.
References (10)
- M.E. May et al.
Arch. Biochem. Biophys.
(1978) - P. Carter
Anal. Biochem.
(1971) - H. Beinert, this series, Vol. 54, p....
- G.B. Tennant et al.
J. Clin. Pathol.
(1969) - L.L. Stookey
Anal. Chem.
(1970)
Cited by (562)
Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe–4S] Fd activity
2024, Journal of Inorganic BiochemistryFerredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1T11I. Electrochemical characterization revealed both Fer1 and Fer1T11I have similar redox transitions (−480 mV and − 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron‑sulfur (FeS) cluster of Fer1. Additionally, disruption of hydrogen bonding around an FeS cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1T11I, which indicate changes to the high spin states and/or spin-spin coupling between the FeS clusters of Fer1. Our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.
Structural and biophysical properties of a [4Fe–4S] ferredoxin-like protein from Synechocystis sp. PCC 6803 with a unique two domain structure
2024, Journal of Inorganic BiochemistryElectron carrier proteins (ECPs), binding iron‐sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp. PCC 6803, the ECP network includes at least nine ferredoxins. Previous research, including global expression analyses and protein binding studies, has offered initial insights into the functional roles of individual ferredoxins within this network. This study primarily focuses on Ferredoxin 9 (slr2059). Through sequence analysis and computational modeling, Ferredoxin 9 emerges as a unique ECP with a distinctive two-domain architecture. It consists of a C-terminal iron‑sulfur binding domain and an N-terminal domain with homology to Nil-domain proteins, connected by a structurally rigid 4-amino acid linker. Notably, in contrast to canonical [2Fe2S] ferredoxins exemplified by PetF (ssl0020), which feature highly acidic surfaces facilitating electron transfer with photosystem I reaction centers, models of Ferredoxin 9 reveal a more neutral to basic protein surface. Using a combination of electron paramagnetic resonance spectroscopy and square-wave voltammetry on heterologously produced Ferredoxin 9, this study demonstrates that the protein coordinates 2×[4Fe4S]2+/1+ redox-active and magnetically interacting clusters, with measured redox potentials of −420 ± 9 mV and − 516 ± 10 mV vs SHE. A more in-depth analysis of Fdx9's unique structure and protein sequence suggests that this type of Nil-2[4Fe4S] multi-domain ferredoxin is well conserved in cyanobacteria, bearing structural similarities to proteins involved in homocysteine synthesis in methanogens.
Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal
2023, Journal of Biological ChemistryThe Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.
Rapid-reaction kinetics of the bifurcating NAD<sup>+</sup>-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus
2023, Journal of Biological ChemistryWe have investigated the kinetics of NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI’s large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD+; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting kred of 36 ± 1.6 s−1 and apparent KdNADPH of 5 ± 1.2 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD+, the system can bifurcate and generate low-potential electrons without NAD+. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD+ demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD+ and ferredoxin. The tight coupling between NAD+ and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.
The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry
2023, Journal of Biological ChemistryMany microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.
First report on the toxicity of SARS-CoV-2, alone and in combination with polyethylene microplastics in neotropical fish
2023, Science of the Total EnvironmentThe COVID-19 pandemic has caused unprecedented negative impacts in the modern era, including economic, social, and public health losses. On the other hand, the potential effects that the input of SARS-CoV-2 in the aquatic environment from sewage may represent on non-target organisms are not well known. In addition, it is not yet known whether the association of SARS-CoV-2 with other pollutants, such as microplastics (MPs), may further impact the aquatic biota. Thus, we aimed to evaluate the possible ecotoxicological effects of exposure of male adults Poecilia reticulata, for 15 days, to inactivated SARS-CoV-2 (0.742 pg/L; isolated SARS.CoV2/SP02.2020.HIAE.Br) and polyethylene MP (PE MPs) (7.1 × 104 particles/L), alone and in combination, from multiple biomarkers. Our data suggest that exposure to SARS-CoV-2 induced behavioral changes (in the open field test), nephrotoxic effect (inferred by the increase in creatinine), hepatotoxic effect (inferred by the increase in bilirubin production), imbalance in the homeostasis of Fe, Ca, and Mg, as well as an anticholinesterase effect in the animals [marked by the reduction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity]. On the other hand, exposure to PE MPs induced a genotoxic effect (assessed by the comet assay), as well as an increase in enzyme activity alpha-amylase, alkaline phosphatase, and carboxylesterases. However, we did not show synergistic, antagonistic, or additive effects caused by the combined exposure of P. reticulata to SARS-CoV-2 and PE MPs. Principal component analysis (PCA) and values from the “Integrated Biomarker Response” index indicate that exposure to SARS-CoV-2 was determinant for a more prominent effect in the evaluated animals. Therefore, our study sheds light on the ecotoxicity of the new coronavirus in non-target organisms and ratifies the need for more attention to the impacts of COVID-19 on aquatic biota.