The internal alternative NADH dehydrogenase (Ndi1) is the electron input in the Saccharomyces cerevisiae respirasome

Complex I is absent in mitochondria from Saccharomyces cerevisiae; instead, three rotenone-insensitive NADH dehydrogenases are present: two on the external (Nde1 and Nde2) and one on the internal (Ndi1) leaf of the inner mitochondrial membrane. In a previous work (1), we reported the presence of a supercomplex in S. cerevisiae constituted by the Ndi1 and complexes III₂ and IV with an apparent MW of 1600 kDa. In this work, respirasomes from WT and NDE1Δ/NDE2Δ strains were isolated, and their activities characterized. Kinetic characterization of NADH:DBQ oxidoreductase activity from respirasomes, as well as free Ndi1, showed V_max values of 0.85 ± 0.01, 0.82 ± 0.02, and 0.51 ± 0.02 μmol NADH oxidized·min^-1·mg^-1 for WT respirasome, NDE1Δ/NDE2Δ respirasome, and free Ndi1, respectively. The kinetic model for WT- and NDE1Δ/NDE2Δ respirasome was a Ping Pong Bi-Bi mechanism with two different stable enzyme forms, free (E) and modified enzyme (F); while the free Ndi1 exhibited a Random Bi-Bi mechanism with the ternary complex NADH-Ndi1-ubiquinone. This suggests that the interaction of Ndi1 with complexes III₂ and IV in the respirasome modifies its kinetic mechanism. Oxygen consumption values were 0.35 ± 0.07 and 0.34 ± 0.07 μmol O₂·min^-1·mg^-1 for WT and NDE1Δ/NDE2Δ respirasomes, respectively. The values for NADH/O₂ ratio were 2.4 ± 1.4 and 2.4 ± 1.6 for WT and NDE1Δ/NDE2Δ respirasomes, respectively, suggesting that electron flux from NADH to oxygen occurs in the S. cerevisiae respirasome. The electron transfer from NADH to oxygen was inhibited by flavone, antimycin A, or cyanide, but the NADH dehydrogenase activity of the respirasome was insensitive to antimycin A or cyanide, indicating that no codependence of respirasomal-Ndi1 activity occurs as reported in the Ustilago maydis respirasome. This result indicates that the activity of respirasomal Ndi1 may contribute to the quinol pool with no evidence of direct substrate channeling. This is the first evidence of the Ndi1 role as the electron input in the respirasome from S. cerevisiae.