Binding and screening by cations and the effect on exogenous NAD(P)H oxidation in Neurospora crassa mitochondria

1. The uncoupled oxidation of exogenous NADH by mitochondria from Neurospora crassa has a pH optimum at 7.0. In the presence of EDTA (1 mM) the optimum is at pH 6.5; maximal inhibition (65%) occurs at pH 7.2. This is comparable to the results with higher plant mitochondria. 2. The corresponding pH optima for NADPH oxidation are 7.75 (control), 7.0 (+ EDTA) and 8.0 (effect of EDTA), respectively. NADPH oxidation is completely inhibited by EDTA at pH 8.0. These pH optima are all about 1 higher than observed in mitochondria from higher plants. 3. The inhibition of NADH oxidation by EDTA is shown to be due to the removal of Mg2+ bound to the mitochondrial membranes. 4. It is shown that 9-aminoacridine can be used to monitor the surface potential of the membranes of Neurospora mitochondria. 5. Cations stimulate NADH oxidation by Neurospora mitochondria in a manner consistent with the theory of the diffuse layer. Quantitatively, the results suggest that Neurospora mitochondria contain fewer charges per mass of protein than Jerusalem artichoke (Helianthus tuberosus) mitochondria but more than mitochondria from Arum maculatum spadices. 6. A good correlation is found between the effect of La3+ on the fluorescence of 9-aminoacridine in the presence of mitochondria and on the oxidation of NADH by the mitochondria. La3+ has different effects on mitochondria from Neurospora, Jerusalem artichoke tubers and Arum spadices. THe results indicate that the fluorescence of 9-aminoacridine can be used to monitor binding sites on biological membranes.