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Insights on the endogenous labile iron pool binding properties

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Abstract

Under normal physiological conditions, the endogenous Labile Iron Pool (LIP) constitutes a ubiquitous, dynamic, tightly regulated reservoir of cellular ferrous iron. Furthermore, LIP is loaded into new apo-iron proteins, a process akin to the activity of metallochaperones. Despite such importance on iron metabolism, the LIP identity and binding properties have remained elusive. We hypothesized that LIP binds to cell constituents (generically denoted C) and forms an iron complex termed CLIP. Combining this binding model with the established Calcein (CA) methodology for assessing cytosolic LIP, we have formulated an equation featuring two experimentally quantifiable parameters (the concentrations of the cytosolic free CA and CA and LIP complex termed CALIP) and three unknown parameters (the total concentrations of LIP and C and their thermodynamic affinity constant Kd). The fittings of cytosolic CALIP × CA concentrations data encompassing a few cellular models to this equation with floating unknown parameters were successful. The computed adjusted total LIP (LIPT) and C (CT) concentrations fall within the sub-to-low micromolar range while the computed Kd was in the 10−2 µM range for all cell types. Thus, LIP binds and has high affinity to cellular constituents found in low concentrations and has remarkably similar properties across different cell types, shedding fresh light on the properties of endogenous LIP within cells.

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Notes

  1. Quadratic equation:

    $$[{\text{CALIP}}]^{2} \;-({\text{K}}_{{{\text{CA}}}}^{{ - 1}} {{ + }}[{\text{CA}}_{{\text{T}}} ] + [{\text{LIP}}_{{\text{T}}} ])[{\text{CALIP}}] + [{\text{CA}}_{{\text{T}}} ][{\text{LIP}}_{{\text{T}}} ] = 0$$

    Solution:

    $$\begin{gathered} \left[ {{\text{CALIP}}} \right] = \left( {{\text{K}}_{{{\text{CA}}}}^{-1} + [{\text{CA}}_{{\text{T}}} ] + [{\text{LIP}}_{{\text{T}}} ]} \right)-{\text{ }}\sqrt[2]{{\left( {{\text{K}}_{{{\text{CA}}}}^{-1} + [{\text{CA}}_{{\text{T}}} ] + [{\text{LIP}}_{{\text{T}}} ]} \right)^{2} -(4}}[{\text{CA}}_{{\text{T}}} \left] {} \right[{\text{LIP}}_{{\text{T}}} ])/2. \hfill \\ \end{gathered}$$

    The second solution returned negative value for KCA.

  2. $$\text{Quadratic equation:}\,[{\text{CALIP}}]^{2} + ({\text{K}}_{{\text{d}}} {\text{K}}_{{{\text{CA}}}} \left[ {{\text{CA}}} \right] + [{\text{C}}_{{\text{T}}} ]-[{\text{LIP}}_{{\text{T}}} ])[{\text{CALIP}}]-{\text{K}}_{{\text{d}}} {\text{K}}_{{{\text{CA}}}} \left[ {{\text{CA}}} \right][{\text{LIP}}_{{\text{T}}} ] = 0.$$
    $$\text{Solution}: \left[ {{\text{CALIP}}} \right] = \frac{{ - ({\text{K}}_{{\text{d}}} {\text{K}}_{{{\text{CA}}}} \left[ {{\text{CA}}} \right] + [{\text{C}}_{{\text{T}}} ]-[{\text{LIP}}_{{\text{T}}} ]) + \sqrt{{({\text{K}}_{{\text{d}}} {\text{K}}_{{{\text{CA}}}} \left[ {{\text{CA}}} \right] + [{\text{C}}_{{\text{T}}} ][{\text{LIP}}_{{\text{T}}} ])^{2} ( - 4{\text{K}}_{{\text{d}}} {\text{K}}_{{{\text{CA}}}} [{\text{CA}}][{\text{LIP}}_{{\text{T}}} ])}}}}{2}.$$

    It was assumed that “free” [LIP] is negligible to the sum of [CLIP] + [CALIP] under the experimental conditions. This is justified by the finding that the hexaaqua Fe(II) complex is not LIP. The second solution returned negative unacceptable values for CT and/or Kd.

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Funding

We acknowledge the financial support from FAPESP (2013/07937-8). JCJT is a member of the Research, Innovation and Dissemination Center (RIDC) Redoxoma (FAPESP).

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  1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, 14040-901, Brazil

    André Luís Condeles, Gabriel Simonetti da Silva, Maria Beatriz Braghetto Hernandes & José Carlos Toledo Junior

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  1. André Luís Condeles
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  2. Gabriel Simonetti da Silva
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  3. Maria Beatriz Braghetto Hernandes
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  4. José Carlos Toledo Junior
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Conceptualization: [JCTJ]; Methodology: [JCTJ, ALC, GSS]; Formal analysis and investigation: [ALC, GSS, MBBH]; Writing, review and editing: [JCTJ]; Funding acquisition: [JCTJ], Supervision: [JCTJ].

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Correspondence to José Carlos Toledo Junior.

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Condeles, A.L., da Silva, G.S., Hernandes, M.B.B. et al. Insights on the endogenous labile iron pool binding properties. Biometals (2024). https://doi.org/10.1007/s10534-024-00591-4

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