Spectroscopic Interrogation of Thiolate Hydrogen Bonding in the CO Sensor Protein CooA: Implications for Thiolate Ligation Stability and Cytochrome P450 Function.

Cysteine (Cys) thiolate coordination in hemoproteins is a unique ligation motif found in enzymes and small molecule sensors. It is posited that divergence between these two functional classes of heme thiolate proteins is a result of distinct hydrogen bonding (H-bonding) interactions with the axial Cys(thiolate) ligand. To further test this hypothesis, we report a spectroscopic analysis of thiolate H-bonding in CooA, a carbon monoxide-sensing heme protein from Rhodospirillum rubrum that is known to switch between Cys(thiolate) and histidine coordination upon reduction. We generated CooA variants with alterations at two residues, Asn and His, which are postulated to influence H-bonding to Cys on the basis of an Fe(II) CooA structure. Using a combination of electronic absorption, electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR) spectroscopies, we identified several CooA variants that exhibit changes in thiolate donor strength and propose an H-bonding model in which Asn orients His for optimal H-bonding with Cys in Fe(III) CooA. Further, we spectroscopically characterize pyrrolidine-bound CYP119, a cytochrome P450, to mimic the first coordination sphere of Fe(III) CooA. Our data unequivocally show that CooA contains a stronger thiolate-Fe bond than the pyrrolidine-bound CYP119, suggesting that Cys(thiolate) H-bonding interactions in CooA are significantly weaker. These results support the hypothesis that thiolate H-bonding is a significant differentiator between the two classes of heme thiolate proteins.