Solvent isotope effects on the structure and function of mitochondrial membranes in aqueous media

The structural stability of mitochondrial membranes and the enzyme complexes of the electron transport system, and the solubility of a small molecular-weight nonelectrolyte (2-methylnaphthoquinone), have been studied as a function of water structure. D₂O, which is considered to be more structured than ordinary water, and H₂O were used as solvents in conjunction with chaotropic ions which have been shown to break down water structure. Assays for membrane stability were (a) resolution with respect to solubilization of at least one constituent enzyme, and (b) chaotrope-induced lipid autoxidation, which is a measure of structural destabilization. Solvent isotope effects expressed as the quotient of chaotrope (NaClO₄) concentration ($\text{C}{}_{\mathrm{<mtext>D</mtext>}}\text{C}{}_{\mathrm{<mtext>H</mtext>}}$) necessary to elicit the same effect were found to be (a) essentially constant for each system over a wide range of NaClO₄ concentration, and (b) limited to the narrow range of 1.2–1.8 in all tests despite significant differences in the systems studied and the measurements used. The magnitude and the constancy of the isotope effects indicate that increased membrane stability (i.e., the increased strength of hydrophobic interactions in membranes), and decreased water-solubility of nonelectrolytes in D₂O are mainly due to the higher degree of order of the deuterated solvent. Thus, in the mitochondrial electron transport chain and many other enzyme systems where solvent isotope effects have been observed, the isotope effect appears to be more a consequence of conformational changes imposed on the enzymes by D₂O, because it is a more structured solvent, rather than an indication of direct involvement of protons or the water molecule in the reaction mechanisms.