Ferritin exhibits Michaelis–Menten behavior with oxygen but not with iron during iron oxidation and core mineralization

Bou-Abdallah, Fadi; Flint, Nicholas; Wilkinson, Tyler; Salim, Samantha; Srivastava, Ayush; Poli, Maura; Arosio, Paolo; Melman, Artem

doi:10.1039/c9mt00001a

Cited by 13 publications

(24 citation statements)

References 31 publications

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“…The iron oxidation kinetics of TgFer, D129A/E132A and E168A mutants were examined by FlexA-200 full-wavelength Microplate reader (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China) at 25°C, as previously described ( Bou-Abdallah et al, 2019 ). Oxygen-free aliquots of Fe 2+ samples (50 mM) were prepared by dissolving Mohr’s salt in 0.1% (v/v) HCl under anaerobic conditions.…”

Section: Methodsmentioning

confidence: 99%

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa

Ming

Jiang

Huo

et al. 2022

Front. Mol. Biosci.

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In addition to its role as an iron storage protein, ferritin can function as a major detoxification component in the innate immune defense, and Cu2+ ions can also play crucial antibacterial roles in the blood clam, Tegillarca granosa. However, the mechanism of interaction between iron and copper in recombinant Tegillarca granosa ferritin (TgFer) remains to be investigated. In this study, we investigated the crystal structure of TgFer and examined the effects of Fe2+ and Cu2+ ions on the TgFer structure and catalytic activity. The crystal structure revealed that TgFer presented a typically 4–3–2 symmetry in a cage-like, spherical shell composed of 24 identical subunits, featuring highly conserved organization in both the ferroxidase center and the 3-fold channel. Structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Cu2+ ions appear to bind preferentially with the 3-fold channel as well as ferroxidase site over Fe2+ ions, possibly inhibiting the ferroxidase activity of TgFer. Our results present a structural and functional characterization of TgFer, providing mechanistic insight into the interactions between TgFer and both Fe2+ and Cu2+ ions.

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Section: Methodsmentioning

confidence: 99%

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa

Ming

Jiang

Huo

et al. 2022

Front. Mol. Biosci.

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“…First, it is possible that the value for the apparent K O2 reflects the value of the intrinsic K O2 . Such a value for K O2 is highly unlikely as this would put DAD in direct competition with important aerobic enzymes including ferritin (K O2 : ~1 μM) [23] and cytochrome c oxidase (K O2 : ~1 μM) [24,25]. Alternatively, the O 2 -dependence could be masked by a subsequent, irreversible step as can be seen in a rapid-equilibrium ordered mechanism: [26][27][28].…”

Section: Substrate-dependence Of the Steady-state Kineticsmentioning

confidence: 99%

The metal- and substrate-dependences of 2,4′-dihydroxyacetophenone dioxygenase

Roberts

Connor

Cavé

et al. 2020

Archives of Biochemistry and Biophysics

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While the enzyme, 2,4′-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4′-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate-and productdependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe 3+ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for k cat and K DHA of 2.4 s −1 and 0.7 μM, respectively, but no significant dependence on the concentration of O 2 . A low-level inhibition (K I = 1100 μM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O 2 to yield an intermediate complex that irreversibly collapses to products.

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“…Iron not immediately utilized by cells is stored in ferritin, a ubiquitous 24-mer iron biomineralizing protein that plays a critical role in iron homeostasis and serves as the principal reservoir of metabolic iron within the cell [ 1 , 2 , 3 , 4 ]. While considerable progress has been achieved towards understanding the ferritin in-vitro iron sequestration mechanisms [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ], iron mobilization is not sufficiently understood and is rather controversial [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. In vitro (or in cell-free systems), the rates of iron uptake and oxidation depend on the concentration of ferritin, its iron content, and the concentration of added Fe(II) cations, but not on the concentration of oxygen [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

Smith

Srivastava

Reutovich

et al. 2022

IJMS

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Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

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Ferritin exhibits Michaelis–Menten behavior with oxygen but not with iron during iron oxidation and core mineralization

Abstract: Iron uptake into mammalian ferritins reveals oxygen (but not iron) saturation kinetics and physiologically relevant Km,O2 values.

Cited by 13 publications

References 31 publications

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa

The metal- and substrate-dependences of 2,4′-dihydroxyacetophenone dioxygenase

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

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