Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Study of manganese binding to the ferroxidase centre of human H-type ferritin Matteo Ardini a,1 , Barry D. Howes b , Annarita Fiorillo a , Elisabetta Falvo a,c , Silvia Sottini b , Donella Rovai b , Marco Lantieri d , Andrea Ilari c , Dante Gatteschi b , Gabriele Spina e , Emilia Chiancone a,c , Simonetta Stefanini a, ⁎ , Maria Fittipaldi e, ⁎ a Department of Biochemical Sciences “A. Rossi-Fanelli”, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy b INSTM, Department of Chemistry “U. Schiff”, University of Florence, Via della Lastruccia, 3-13 50019 Sesto Fiorentino, Florence, Italy c CNR-Institute of Molecular Biology and Pathology, P.le Aldo Moro 5, 00185 Rome, Italy d ISC-CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Florence, Italy e INSTM, Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Florence, Italy ARTICLE INFO Keywords: Human H ferritin Ferroxidase centre Heterodimetal manganese-iron centre Mössbauer EPR ABSTRACT Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been in- vestigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV–Vis ferroxidation kinetics, fluorimetric titrations, multi- frequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron. 1. Introduction Manganese is a metal essential for human health. It is an important cofactor in many different enzymes involved in the metabolism of carbohydrates, amino acids, and cholesterol as well as in those acting at the forefront against oxidative stress such as superoxide dismutase. Further, manganese is also required for proper iron metabolism and, thus, plays an important role in anaemia prevention [1]. Among biologically active metals, both iron (Fe) and manganese (Mn) are of crucial importance, primarily for their role in enzyme- catalysed redox chemistry. Their chemical properties suggest that they are interchangeable, as their ligand preferences and binding sites in many enzymes are very similar. The biochemical choice of Mn rather than Fe is often imposed by different functional requirements and, in such cases, generally facilitates the enzyme activity [2]. However, some evidence suggests that iron and manganese can share common ab- sorption and transport mechanisms [3]. Moreover, it is important to recall that iron deficiency has been shown to increase the risk of manganese accumulation in the brain that may lead to the development of adverse neurological effects [4]. A heterodimetal Mn-Fe cofactor has been found in ribonucleotide reductase (RNR), a protein belonging to the ferritin (Ft) superfamily, endowed with a carboxylate-bridged di-iron centre localized within the 4-helix bundle, which uses the Mn-Fe complex for the reduction of nucleotides to deoxynucleotides in all organisms [5–7]. Recently, a discrimination mechanism between Fe and Mn at the specific binding sites has been proposed in the GkR21ox1 protein, a homolog to the RNRs family from the thermophile bacterium Geobacillus kaustophilus, and shown to be correlated to the protein structure and oxygen (O 2 ) binding properties [7]. In this regard, the natural presence or artificial formation of mixed Mn-Fe cofactors can be postulated for other ferritin- related proteins with an active site able to oxidize Fe(II) in the presence of O 2 . Recently, the formation of an Fe(II)-Mn(II) heterodimetal fer- roxidase centre has been identified in another ferritin-related protein, https://doi.org/10.1016/j.jinorgbio.2018.02.003 Received 16 October 2017; Received in revised form 11 January 2018; Accepted 4 February 2018 ⁎ Corresponding authors. 1 Istituto Italiano di Tecnologia, Via Morego 16, 16136 Genova, Italy. E-mail addresses: simonetta.stefanini@uniroma1.it (S. Stefanini), maria.fittipaldi@unifi.it (M. Fittipaldi). Journal of Inorganic Biochemistry 182 (2018) 103–112 Available online 09 February 2018 0162-0134/ © 2018 Elsevier Inc. All rights reserved. T