The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conservedputative heme-binding proteins that contains a number of members in animal, plant and bacterial spe-cies. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL,have been determined in 2006 and 2011 respectively.In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme boundmurine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic innature, was thought to also involve electrostatic interactions between heme propionate groups and pos-itively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting inKdvalues in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMRrelaxation time measurements for human HEBP1 describe a rigid globular protein with no change inmotional regime upon heme binding.X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the newheme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 Å). Results from a HSQC spectrum cen-tred on the histidine side chain Nd-proton region for HEBP2 confirm that HEBP2 does not bind heme viaH42 as no chemical shift differences were observed upon heme addition for backbone NH and Ndprotons.A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range ofcellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammalsand a potential link between heme release under oxidative stress and human HEBP1 is also examinedusing recent data. However, at the present moment, trying to relate function to the involvement of hemehttps://doi.org/10.1016/j.ccr.2021.2141890010-8545/Ó2021 Elsevier B.V. All rights reserved.Abbreviations:HEBP1, Heme-binding protein 1 (or p22HBP); HEBP2, Heme-binding protein 2 (or SOUL); BH3, Bcl-2 homology 3; HSQC, heteronuclear single quantumcoherence spectroscopy; TROSY, transverse relaxation optimized spectroscopy; FQ, fluorescence quenching; PPIX, protoporphyrin IX; sGC, solubleGuanylyl Cyclase; hetNOE,heteronuclear nuclear Overhauser effect; rmsd, root mean squared deviation; BMRB, biological magnetic resonance bank; VAST, vector alignment search tool; MEL, murineerythroleukemia.⇑Corresponding authors.E-mail addresses:brian.goodfellow@ua.pt(B.J. Goodfellow),mdam@fct.unl(A.L. Macedo).Coordination Chemistry Reviews 448 (2021) 214189Contents lists available atScienceDirectCoordination Chemistry Reviewsjournal homepage: www.elsevier.com/locate/ccr