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A quantitative method has been developed to analyze the amino acid composition of protein subunits directly from the Coomassie Blue-stained band of polyacrylamide gel columns after electrophoresis. It is an improved method originally reported by Houston (Houston, L. L. (1971) Anal. Biochem. 44, 81--88). The results obtained can be thus used for the calculation of the molar ratios of subunit components of protein. The manipulation of the method and computation of the results are illustrated by a very complicated lipoprotein complex. The subunit molar ratios of the reconstitutively active cytochrome b-c1 complex were determined to be 2, 2, 2, 3, 2, 2, and 5 among the seven bands of the corresponding molecular weights of 53 000, 50 000, 37 000, 30 000, 28 000, 17 000, and 15 000, from gel electrophoretic columns. The amino acid composition of each subunit fraction determined directly from hydrolysis of gel was comparable with that obtained by actual isolation of each subunit.

EPR spectra of nitric oxide complexes of cytochrome c oxidase were studied as a function of temperature in the fully reduced form and in a mixed valence form. In the 12-77 K range, temperature-dependent motion changed the EPR spectral line shape of the mixed valence form but not the fully reduced form. Two separate hypotheses which both indicate cytochrome a-cytochrome a3 interaction could account for this phenomenon: i.e. either, in response to redox changes at cytochrome a, a changed ligand binding environment occurs at cytochrome a3 which has conformations that down to 40 K can interconvert on a 10(8) s-1 time scale, or dipolar spin-spin interaction occurs between the NO-liganded a3 center and a paramagnetic metal center in cytochrome a. This interaction is then modulated by the temperature-dependent spin-lattice relaxation behavior of the center in cytochrome a. If this latter hypothesis is correct, the intercenter distance can be estimated at 15 A, and the more likely interacting center can be predicted to be the heme moiety of cytochrome a.