Binding of spin-labeled carboxyatractylate to mitochondrial adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier as studied by electron spin resonance

Abstract

The spin-label 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid was attached to the inhibitor carboxyatractylate of the mitochondrial ADP/ATP carrier. Being closely linked to the inhibitor, the spin-label should reflect the mobility of the carboxyatractylate. When bound to the carrier in mitochondria, spin-labeled carboxyatractylate reveals a most unusual hyperfine splitting of 72 G. A second spectral component with a hyperfine splitting of 62 G is also mainly due to carrier-bound inhibitor. A similar spectrum with somewhat reduced hyperfine splitting was observed with the detergent-solubilized protein, whereas reincorporation into phospholipid membranes yielded almost the same spectra as in mitochondria. The carrier-bound spin-label is concluded to be highly immobilized. The less immobilized spectral component is discussed in terms of strongly anisotropic label motion. In addition, the unusual splitting is interpreted to indicate the highly polar environment of the nitroxide. The interpretations are supported by the temperature dependence, which indicates a reversible progressive spin-label mobilization up to 50 degrees C. Membrane-impermeable reducing agents showed that the spin-label is easily accessible from the aqueous phase.