Neutral thiol as a proximal ligand to ferrous heme iron: implications for heme proteins that lose cysteine thiolate ligation on reduction.

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O(2). A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175CD235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.