Structure and Mechanism of Mammalian Stearoyl‐CoA Desaturase‐1 and its Redox Partners
Authors: Shen Jiemin , Wu Gang , Tsai Ah‐Lim , Zhou Ming 
Published: May 2022

Mammalian stearoyl‐CoA desaturase‐1 (SCD1) is a membrane‐embedded diiron enzyme that introduces the first double‐bond to a saturated long‐chain fatty acid (in the form of an acyl‐CoA). Structures of mouse and human SCD1 were reported previously, however, these studies found that the heterologously expressed mammalian SCD1 contains two zinc ions (Zn2+), likely mis‐incorporated during the expression, and that the enzyme is inactive. We developed a protocol to enhance iron incorporation and obtained fully functional SCD1. We also solved the structure of mouse SCD1 with a diiron center. The diiron center in SCD1 has an Fe‐Fe distance of 6.4 Å and are coordinated entirely by histidine residues. This configuration is different from all previously characterized diiron centers, in which carboxylate/oxygen groups bridge two irons and the Fe‐Fe distance is usually less than 4 Å. The longer distance and different coordination of the diiron center in SCD1 prompted us to further examine its redox chemistry.

The redox cycle of the diiron center in SCD1 requires reducing equivalents from NADH and the electrons are transferred via cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5), both of which have a single transmembrane (TM) anchor. Although proximity of these proteins is expected, it is not clear how b5R, cyt b5and SCD1 could interact on membranes. We showed that b5R, cyt b5 and SCD1 form a ternary complex in cells, and that the complex is stabilized mainly by interactions of the TM domains. We also showed that b5R and cyt b5 form stable binary complex, and so are cyt b5 and SCD1. These results will enable further structural and functional analyses to understand the mechanism of electron transfer in SCD1, and since b5R and cyt b5are partners to several other redox enzymes, our discoveries of stable binary and ternary complex may also be applicable in these enzymes as well.

We assembled purified b5R, cyt b5 and SCD1 proteins in a test tube and enabled in‐vitro study of SCD1’s activity. We found that SCD1 loses activity within ten turnovers. This deactivation is due to the loss of a single iron ion in the diiron center. Addition of ferrous ions in the reaction mixture prevents such deactivation. These results suggest that structural changes during the enzymatic cycle is significant enough to weaken the coordination of one of the iron ions. We then measured electron paramagnetic resonance (EPR) spectra of SCD1, and found strong signals around g = 4 – 6 from high spin iron(s), and a broad symmetric feature centered at g = 2, which may be due to exchange‐ or dipolar‐coupled ferric irons. The broad feature at g = 2 disappeared in SCD1 mutants that disrupt one of the iron binding sites, and in the wild‐type SCD1 devoid of an iron after reaction cycles. Although further experiments are required to interpret and understand the EPR spectra, these results helped identify the iron prone to dissociation during the reaction cycle. In summary, results from this study enhanced our knowledge of the diiron center in SCD1, and established an avenue of research for understanding the redox chemistry of the novel diiron center in SCD1.