Rational design of tertiary coordination sphere of a heme-based sensor for two-orders enhanced oxygen affinity

Abstract

Biological O₂ sensing is crucial for diverse physiological functions across all forms of life. Heme-containing proteins achieve this by binding O₂ to their iron center and have been found to display O₂ affinities spanning several orders of magnitude. Despite decades of investigation into the structure and function of heme-based O₂ sensors, the molecular mechanisms that enable the tuning of O₂ affinity to match specific physiological roles remain unclear. Here, we utilize the O₂ sensing mycobacterial DosS protein as a model system to explore the role of heme iron's tertiary coordination sphere in controlling its O₂ affinity. By rationally and systematically modifying the tertiary coordination sphere to promote the formation of a Trp-Tyr-Asn H-bond triad within the heme's distal pocket, we have enhanced the O₂ affinity of WT DosS by over 150-fold. The rationally designed DosS exhibited a K _d value of 3 ± 1 nM, compared to 460 ± 80 nM for WT DosS. Employing a combination of structural, biochemical, spectroscopic, and computational studies, our analysis of WT and designed DosS variants highlights how the interplay between distal H-bond networks and heme-pocket electrostatics drives large differences in their O₂ sensing capabilities. Ultimately, our work shows how metalloenzymes can dramatically alter their sensitivity to diatomic signaling molecules by tuning the tertiary coordination sphere, broadly impacting how we understand related biological sensing and signaling.

Fig. 1:

a) Schematic shows O₂ binding to ferrous heme. The partial negative charge on iron-bound O₂ is stabilized via H-bonding to SCS tyrosine residue. The graph shows the range of Kd value spanned by heme-based O₂ sensors that feature tyrosine in their SCS (in gray). The cyan bar represents the Kd value spanned by DosS sensors designed in this work that feature tyrosine in their SCS. b) Crystal structure of ferric form of Cs H-NOX that zooms into its distal heme pocket. H-bonding distances are shown via dashed lines. c) Crystal structure of ferric form of WT, d) F98W, and e) E87N GAF-A DosS that zooms into their distal heme pocket. H-bonding distances are shown via dashed lines. Blue structure in d-e show corresponding SCS/TCS residues in WT GAF-A DosS overlaid.

Fig. 2:

a) A distal heme pocket zoomed-in snapshot from MD simulations of O₂-bound NWL GAF-A DosS. H-bonding distances are shown via dashed lines. UV-Vis spectroscopic studies showing ferrous forms of b) WT and c) NWL DosS (in gray) binding to NO (in blue). Inset in b) shows the structure of a 6c NO/His bound ferrous heme and c) shows the structure of a 5c NO bound ferrous heme with proximal histidine dissociated from the heme iron.

Fig. 3:

a) UV–Vis spectral changes in WT DosS upon binding O₂ at various free O₂ concentrations measured using the optode. b) Difference spectra showing spectral changes when WT DosS binds O₂. c) O₂ affinity plots for WT (dark blue), E87N (purple), and NWL (maroon) DosS proteins (n = 3).

Fig. 4:

a) Electrostatic potential maps of a) WT and b) NWL DosS obtained using their DFT simulated heme bound to O₂ structures. Histogram depicting distances sampled between c) N87’s amine hydrogens and Y171’s hydroxyl oxygen, d) W98’s N1 hydrogen and Y171’s hydroxyl oxygen and e) W98’s N1 hydrogen and distal oxygen atom of heme-bound O₂ in MD simulated structure of O₂-bound NWL DosS. Dashed line in c-e represents cut-off of H-bonding distances at 3.5 angstrom.