Modeling proline ligation in the heme-dependent CO sensor, CooA, using small-molecule analogs.

CooA, the only protein known to employ proline as a heme ligand, is a CO-activated transcription factor found in the bacterium Rhodospirillum rubrum. Proline is a heme ligand in both the Fe(III) and Fe(II) states; the sixth ligand is cysteinate in Fe(III) CooA and histidine in Fe(II) CooA. When CO binds to Fe(II) CooA, it selectively replaces the proline ligand, activating the protein. The proposed roles of proline are to stabilize the heme pocket during the redox-mediated ligand switch and to form a weak metal-ligand bond that is preferentially cleaved to bind CO. To explore this latter proposal, binding affinity, structural, and density functional theory computational studies were performed using pyrrolidine and 2-methylpyrrolidine as analogs of proline, and imidazole as an analog of histidine. Measurement of the binding properties of these amino acid analogs in two different protein environments, CooA variant deltaP3R4 and myoglobin, revealed that CooA is tailored to accept the bulky proline ligand. Furthermore, the high pKa of proline facilitates selective replacement by CO. Model metalloporphyrin X-ray and computational structures suggest that the key factor leading to lengthening of the Fe-ligand bond and decreased binding affinity is steric hindrance at the C-2 position of the pyrrolidine ring. These data afford a more complete understanding of how CooA utilizes the weak proline ligand to direct CO to the distal position, thus ensuring selective retention of the histidine ligand.