Entrance of the proton pathway in obb_3-type heme-copper oxidases

摘要

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O_2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb_3) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 [Rhodobacter sphaeroides cbb_3 numbering) in the accessory subu-nit CcoP (E25~P). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb_3 by O_2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gin, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25~P as the entry point to the proton pathway in cbb_3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O_2 reduction. The cbb_3 is in addition to O_2 reduction capable of NO reduction, an activity that was largely retained in the E25~P variants, consistent with a scenario where NO reduction in cbb_3 uses protons from the periplasmic side of the membrane.