The mitochondrial oxoglutarate carrier: structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling

The structural and dynamic features of the fourth transmembrane segment of the mitochondrialoxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance(EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutiveresidues from S184 to S201 which are believed to form the transmembrane segment IV were substitutedindividually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutantsexhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined byEPR as a function of the incident microwave power in the presence and absence of two paramagneticperturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. Theperiodicity of the sequence-specific variation in the spin-label mobility and the O2 accessibility parametersunambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier asan R-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicatingthat the helix is amphipatic, with the hydrophilic face containing the residues found to be important fortransport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed,as indicated by the values of normalized mobility, which also suggest that the conformational changesoccurring during transport probably involve the N-terminal region of the helix.