# Iron(III) Complexation by Rhizoferrin, a Fungal Siderophore

Sylvie Grunenwald, Sylvie Blanc, Natacha Rochel, Günther Winkelmann, A.M. Albrecht-Gary

► To cite this version:

Sylvie Grunenwald, Sylvie Blanc, Natacha Rochel, Günther Winkelmann, A.M. Albrecht-Gary. Iron(III) Complexation by Rhizoferrin, a Fungal Siderophore. Quatrièmes Rencontres du GDR 1154 Transport du fer dans les systèmes biologiques. De la chimie de coordination à la biologie moléculaire et cellulaire, Jan 1998, Seillac, France. hal-05367382

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# Iron(III) Complexation by Rhizoferrin, a Fungal Siderophore

S. Grunenwaldª, S. Blancª, N. Rochelª, G. Winkelmannᵇ, A.-M. Albrecht-Garyª

ª) Laboratoire de Physico-Chimie Bioinorganique, UMR 7512 du CNRS, Faculté de Chimie, 1 rue Blaise Pascal, 67000 Strasbourg, France

ᵇ) Microbiology and Biotechnology, University of Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Allemagne

## Introduction

Rhizoferrin is a polycarboxylate siderophore excreted by the pathogenic fungi which belong to *Rhizopus* family¹,³. These fungi are responsible for mucormycosis and their virulence is enhanced by medical treatment with Desferal⁴,⁵ (deferriferrioxamine B, Ciba-Geigy). For a better understanding of the development of these fungi which requires iron uptake outside the cells and release inside, we have carried out thermodynamic, electrochemical and kinetic studies which point out the major parameters involved in the coordination and transport of iron by rhizoferrin under physiological conditions.

## Thermodynamics

### Protonation

<figure><img src="image_3.png" /><figcaption>Solvent: water; I = 2.0; T = (25.0 ± 0.2)°C</figcaption></figure>

<table><thead><tr><th>Ligands</th><th>pK<sub>1</sub></th><th>pK<sub>2</sub></th><th>pK<sub>3</sub></th><th>pK<sub>4</sub></th></tr></thead><tbody><tr><td>glutaric acid</td><td>4.84</td><td>4.09</td><td></td><td></td></tr><tr><td>tartric acid</td><td>3.93</td><td>2.79</td><td></td><td></td></tr><tr><td>of tricarboxylic acid</td><td>13</td><td>5.7</td><td>4.32</td><td>2.92</td></tr></tbody></table>

The pK values were attributed by comparison with carboxylic acids

### Ferric Complexes

<figure><img src="image_4.png" /><figcaption>Solvent: water; I = 2.0; T = (25.0 ± 0.2)°C</figcaption></figure>

<table><thead><tr><th>Coordination Sites</th><th>Ligands</th><th>pFe(III)</th></tr></thead><tbody><tr><td>3 catecholates</td><td>Enterobactin</td><td>35.5</td></tr><tr><td>3 hydroxamates</td><td>Ferrioxamine B</td><td>26.6</td></tr><tr><td>2 hydroxamates<br>1 hydroxyacid</td><td>Aerobactin</td><td>23.3</td></tr><tr><td>2 carboxylic acids</td><td rowspan="2">Rhizoferrin</td><td rowspan="2">19.7</td></tr><tr><td>2 hydroxyacids</td></tr></tbody></table>

$$pFe(III) = -\log [Fe^{3+}] \text{ with } [L]_{\text{tot}} = 10^{-5} \text{ M; } [Fe]_{\text{tot}} = 10^{-6} \text{ M; } p[H] = 7.4$$

Rhizoferrin ferric complexes are less stable than catecholates or hydroxamates siderophores

## Electrochemistry

<figure><img src="image_5.png" /></figure>

$$E_{\text{redox}}(V) = E_{\text{0}} + 0.059 \log \frac{[\text{Fe(III)}]_{\text{L}}[1 + K_{\text{[Fe(III)}]_{\text{L}}}[\text{H}^{+}] + K_{\text{[Fe(III)}]_{\text{L}}}] + K_{\text{[Fe(III)}]_{\text{L}}}[\text{H}^{+}]^{2}]}{[\text{Fe(II)}]_{\text{L}}[1 + K_{\text{[Fe(II)}]_{\text{L}}}[\text{H}^{+}] + K_{\text{[Fe(II)}]_{\text{L}}}] + K_{\text{[Fe(II)}]_{\text{L}}}[\text{H}^{+}]^{2}}}$$

$$K_{\text{Fe(II)}\text{LH}} = (3 \pm 1) \times 10^{-8} \text{ M}^{-1}$$

$$K_{\text{Fe(II)}\text{LH}_2} = (8 \pm 1) \times 10^{-8} \text{ M}^{-1}$$

$$\text{Log } K_{\text{Fe(II)}} = 3 \pm 1$$

<figure><img src="image_6.png" /></figure>

The reduction potential of ferric rhizoferrin at p[H] = 7 is similar to

those of carboxylic complexes and less negative than those of

ferrioxamine B and enterobactin ferric complexes

## Kinetics

### Iron(III) Uptake Mechanism

<figure><img src="image_7.png" /></figure>

$$\begin{array}{l} \text{LH}_6 + \text{FeOH}^{2+} \xrightarrow{k_1} \text{LH}_5\text{Fe} + \text{H}_2\text{O} \\ \text{H}^+\uparrow\downarrow \\ \text{LH}_5 + \text{FeOH}^{2+} \xrightarrow{k_2} \text{LH}_6\text{Fe} + \text{OH}^- \end{array}$$

<table><thead><tr><th>Ligands</th><th>k<sub>f</sub> (FeOH<sup>2+</sup>) (M<sup>-1</sup>s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>hydroxamic acids</td><td>(2.3 to 5) x 10<sup>3</sup></td></tr><tr><td>substituted cathecolates</td><td>(1.5 to 3.1) x 10<sup>3</sup></td></tr><tr><td>deferriferrioxamine B</td><td>3.5 x 10<sup>3</sup></td></tr><tr><td>hydroxyacids</td><td>(3.6 to 6.1) x 10<sup>3</sup></td></tr><tr><td>rhizoferrin LH<sub>6</sub></td><td>(1.3 ± 0.3) x 10<sup>3</sup></td></tr><tr><td>rhizoferrin LH<sub>5</sub></td><td>(5 ± 1) x 10<sup>4</sup></td></tr></tbody></table>

The rate determining step for FeO<sup>ff</sup> is the first water molecule release from the coordination sphere of the metal: Eigen-Wilkins mechanism

### Iron(III) Release Mechanism

<figure><img src="image_8.png" /></figure>

$$\text{Solvent: water; T = 25.0°C; I = 2; } [\text{LF}\text{e}^-] = 6.6 \times 10^5 \text{ M; } [\text{H}^+] = 0.8 \text{ M; } \lambda = 330 \text{ nm; I = 1 cm.}$$

$$\text{First step :} \quad \text{LFe}^{3-} + 6\text{H}^{+} \xrightarrow{\text{fast}} \text{LH}_5\text{Fe}^{2+}$$

$$\text{Second step :} \quad \text{LH}_5\text{Fe}^{2+} \xrightarrow{\text{fast}} \text{LH}_5\text{Fe}^{2+ *}$$

$$\text{Third step :} \quad \text{LH}_5\text{Fe}^{2+ *} + \text{H}^{+} \rightarrow \text{LH}_6 + \text{Fe}^{3+}$$

Two rates limiting steps

## Conclusion

* Under physiological conditions the rhizoferrin ferric complexes are at least 6 orders of magnitude less stable than tri-hydroxamate Fe(III) species

* The reduction of the ferric rhizoferrin complexes is accessible to biological reductants

* The chelation mechanism of iron(III) under acidic conditions is a fast process

* Release mechanism: solwest steps related to the dissociation of the final carboxylic group

## Perspectives

* Spectrophotometric determination of the intermediate complexes by diode arrays stopped-flow measurements

* Determination of the activation parameters

## References

1) Drechsel, H.; Metzger, J.; Freund, S.; Boelaert, J.R.; Winkelmann, *BioMetals* 1991, *4*, 238. 2) Carrano, C.J.;

Drechsel, H.; Kaiser, D.; Jung, G.; Matzanke, B.; Winkelmann, G.; Rochel, N.; Albrecht-Gary, A.-M. *Inorg. Chem*, 1996,

35, 6429. 3) Drechsel, H.; Jung, G.; Zähner, H.; Winkelmann, G. *BioMetals* 1992, *5*, 141. 4) Boelaert, J.R.; de Locht, M.;

Schneider, Y.J.J. *Infect. Dis.* 1994, *169*, 231. 5) Boelaert, J.R.; de Locht, M.; Van Cutsem, *Rev. Med. Microbiol.*

1993, *4*, 171.