Keynote Talk 21st International Conference on Biological Inorganic Chemistry 2025

CooA forms a variable hydrogen bonding network involving its proximal thiolate ligand: comparison to cytochrome P450 and implications for the ligand switching heme proteins (124765)

Judith N Burstyn 1 , Brian R Weaver 1 , Matthew R Dent 2 , Richard J Jodts 3 , Brian M Hoffman 3 , Joshua Telser 4
  1. University of Wisconsin-Madison, Madison, WISCONSIN, United States
  2. Chemistry, Wayne State University, Detroit, Michigan, USA
  3. Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois, United States
  4. Biological, Physical, and Health Sciences, Roosevelt University, Chicago, Illinois, United States

Cysteine (Cys) thiolate coordination in hemoproteins is found in enzymes and in small molecule sensors. A hypothesis posits that functional divergence of these two classes of heme thiolate proteins results from distinctive hydrogen bonding interactions with the axial Cys(thiolate) ligand. To interrogate this hypothesis, we undertook a spectroscopic analysis of thiolate H-bonding in CooA, a carbon monoxide-sensing heme protein from Rhodospirillum rubrum that switches between Cys(thiolate) and histidine coordination upon reduction. Through analysis of a crystal structure of reduced, Fe(II) RrCooA, we predicted that two residues, Asn42 and His77, make key hydrogen bonding contacts in the pocket surrounding Cys75. We generated CooA variants and collected electronic absorbance, EPR, and ENDOR spectra, with the goal of interrogating the effect amino acid changes at positions 42 and 77 on the hydrogen bonding environment of the thiolate ligand. Assessment of protein spectra suggests that there are two distinct hydrogen bonding configurations surrounding Cys75, and we identified several CooA variants that appear to exhibit changes in thiolate donor strength. We propose a model in which Asn42 orients His77 for optimal H-bonding with Cys75 in Fe(III) CooA. In comparing the ENDOR spectra of WT and variant CooA proteins with those of a pyrrolidine-ligated cytochrome P450 (CYP119), which mimics the first coordination sphere of N-terminally Pro-coordinated CooA, it appears that the relative thiolate donor strengths in CooA and P450 differ significantly. Our data imply that CooA contains a much stronger thiolate-Fe bond than pyrrolidine-bound CYP119, suggesting that the Cys(thiolate) H-bonding interactions in CooA are weaker and lending support to the hypothesis that thiolate H-bonding is a differentiator between the ligand-switching sensor proteins (like CooA) and the small molecule activating proteins (like P450).

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  2. Dent, M. R.; Milbauer, M. W.; Hunt, A. P.; Aristov, M. M.; Guzei, I. A.; Lehnert, N.; Burstyn, J. N. Electron Paramagnetic Resonance Spectroscopy as a Probe of Hydrogen Bonding in Heme-Thiolate Proteins. Inorg. Chem. 2019, 58 (23), 16011–16027. https://doi.org/10.1021/acs.inorgchem.9b02506.