Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the
hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood. Crystallographic investigations have shown that the trHb fold (a two-on-two alpha-helical sandwich related to the
globin fold) hosts a
protein matrix tunnel system offering a potential path for
ligand diffusion to the
heme distal site. The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure. Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under
xenon pressure leads to binding of
xenon atoms at specific (conserved) sites along the
protein matrix tunnel. The crystallographic results are in keeping with data from molecular dynamics simulations, where a
dioxygen molecule is left free to diffuse within the
protein matrix. Modulation of
xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles. In a parallel crystallographic investigation on M.
tuberculosis trHbN, we show that
butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the
xenon binding experiments. These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity
ligands/molecules within group I trHbs.