Fig. 4. SCD1 and full-length cyt b5 can form a stable complex with faster electron transfer kinetics.

a Schematic diagram of SCD1-cyt b₅ fusion constructs. b SEC profile (left), SDS-PAGE image (inset), and UV-Vis spectra (right) of the linker-cleaved (magenta) and tethered (black) fusion of SCD1-cyt b₅. Almost identical SEC profile and optical spectra suggest the stable assembly between SCD1 and cyt b₅. c Biphasic kinetics of NADH consumption by b₅R with SCD1-cyt b₅ fusion in the presence of substrate stearoyl-CoA (red) compared to the slow linear decrease of NADH in the absence of substrate (orange). The absorbance of NADH at 340 nm (A₃₄₀) was measured and the y axis is the normalized A₃₄₀ against the initial values (A_{340, 0}). Excess NADH was used in the measurements. d The accelerated electron transfer between reduced cyt b₅ and SCD1 in the SCD1-cyt b₅ complex (brown) compared to that between the individual cyt b₅ and SCD1 (yellow) and auto-oxidation of cyt b₅ (orange). The Soret absorbance of reduced cyt b₅ at 423 nm was monitored. One molar equivalent of NADH was added, which resulted in the initial rising phases of the fast electron transfer to cyt b₅ via b₅R. Rate constants (k) are denoted as mean ± SEM calculated from three independent repeats (n = 3).