Chaotropic resolution of high molecular weight (Type I) NADH dehydrogenase, and reassociation of flavin-rich (Type II) and flavin-poor subunits

Abstract

1. 1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO 4. NADH slows the dissociation. On subsequent stepwise addition of (NH 4) 2SO 4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO − 4 and SO 2 − 4, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. 2. In consequence, the aggregates of subunits that are separated by (NH 4) 2SO 4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH 4) 2SO 4 is flavinpoor, that remaining soluble at 55% satd. (NH 4) 2SO 4 flavin-rich and those separating between 27 and 55% satd. (NH 4) 2SO 4 intermediate in composition. 3. 3. The fraction remaining soluble at 55% satd. (NH 4) 2SO 4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH 4) 2SO 4 contained an additional small subunit (12 kildaltons) and four additional Fe and acid-labile S atoms per protomer. The sedimentation coefficients ( s 0 20, w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH 4) 2SO 4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is co-operative. NADH favours the association of the subunits. 6. 6. Association of the subunits is accompanied by a 10-fold increase in k 2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10 4-fold decrease of the accessibility of ferricytochrome c. The K s (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k 2 is still much less than in the latter enzyme.