Reaction of S. cerevisiae mitochondria with ligands: Kinetics of CO and O2 binding to flavohemoglobin and cytochrome c oxidase

Abstract

Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (CytcO) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, CytcO and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with τ≅270ms, which is significantly slower than observed with the cytosolic yHb (main component τ≅1.3ms). The data indicate that the yHb populations in the different cell compartments differ in structure.

Splitting of the O–O bond at the heme-copper catalytic site of respiratory oxidases

“…The flow-flash measurements were performed using a modified stopped-flow setup (Applied Photophysics) ( 65 , 66 ). Briefly, the ba 3 CytcO was mixed at a ratio of 1:5 with oxygen-saturated buffer [100 mM Hepes and 0.05% DDM (pH 7) or 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and 0.05% DDM (pH 10)].…”

Isolation of yeast complex IV in native lipid nanodiscs

“…For time-resolved kinetic experiments (see below) the mitochondria were prepared devoid of their outer membrane according to [51]. Briefly, mitochondria after running the sucrose CytcO-SMA native discs were stored at 4 0 C.…”