Human radical S-adenosylmethionine domain-containing 1 (RSAD1) is a Heme-binding protein

Abstract

Human radical S-adenosyl-l-methionine domain-containing 1 (hRSAD1) is a recently discovered mitochondrial protein that plays an important yet not fully understood role in cellular function. hRSAD1 belongs to the large and diverse radical S-adenosyl-l-methionine (SAM) superfamily of enzymes that utilize a redox-active [4Fe-4S] cluster and SAM to initiate radical catalysis. In addition, hRSAD1 harbors a putative heme-binding domain. hRSAD1 was expressed in E. coli and purified to homogeneity. The purified hRSAD1 was reconstituted with a [4Fe-4S]²⁺ cluster that could be reduced to the [4Fe-4S]⁺ state, and was characterized using UV–visible and EPR spectroscopy. The ability of hRSAD1 to bind porphyrins was evaluated, revealing that protoporphyrin IX (PPIX) and its metal analogs, including Fe^(II)-PPIX, Fe^(III)-PPIX, and Zn^(II)-PPIX, bind to the reconstituted hRSAD1-[4Fe-4S] protein. The association constant (K_A) for Fe^(III)-PPIX was determined using UV–visible and fluorescence spectroscopy to be (1.6 ± 0.3) × 10⁶ M⁻¹. Additionally, the hRSAD1–[4Fe-4S]–heme complex binds oxygen, carbon monoxide, and cyanide. These findings suggest that hRSAD1 may play a significant role in heme-related metabolic processes.