In biological organisms the ability to sense the local environment and respond accordingly is of paramount importance. A newly discovered category of heme-thiolate proteins has been identified which displays a wide range of functions, from sensing CO to regulating circadian rhythms. Included in this group are six-coordinate heme proteins which are regulated by a ligand switch from the cysteine thiolate ligand to a neutral ligand upon reduction of the heme iron. However, the exact mechanism for the ligand switch is unknown. In previous research a series of iso-1-cytochrome c variants were generated which contained a Met80 to Cys mutation to create the desired thiolate-ligated heme. These variants also contained mutations which introduced potential histidine ligands at various locations to permit a ligand switch identical to the ones in known heme-thiolate proteins. A control variant was made with only the Met80 to Cys mutation. The model protein successfully switched ligands, but so did the control. Other mutations were made which eliminated native histidine residues in order to eliminate possible ligands in the control, but this ultimately could not prevent the ligand switch. The new heme ligand in the Fe2+state is still unknown at this time. Other possible ligands include the N-terminus and oxygens in the protein backbone, which cannot be removed by mutation. The goal of the project has shifted to trying to control the rate of the model protein's ligand switch. The current model's ligand switch occurs in a matter of seconds. Changing the tyrosine at position 76 into either a lysine or an arginine would introduce a positive charge near the negative cysteine residue in our model, potentially slowing the ligand switch, like seen in cystathionine beta-synthase. The results of these mutations and their effects on the protein will be discussed.

Attempts to tune the ligand switch in heme-thiolate model proteins using second sphere ligand effects

247th National Meeting (2014)

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