PubMed · 4338418
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D E Green. 1972-06-20. Membrane proteins: a perspective.. https://pubmed.ncbi.nlm.nih.gov/4338418/
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The substrate for the hydrolytic activity of membrane-bound pyrophosphatase is the PP(i)-Mg2+ complex. The enzyme has no activity when the free Mg2+ concentration is lower than 10 microM (at 0.5 mM-PP(i)-Mg2+), and therefore free Mg2+ is an essential activator of the hydrolytic activity. The Km for the substrate changes in response to variation in free Mg2+ concentration, from 10.25 to 0.6 mM when free Mg2+ is increased from 0.03 to 1.0 mM respectively. The Km for Mg2+ depends on the substrate concentration: the Km decreases from 0.52 to 0.14 mM from 0.25 to 0.75 mM-PP(i)-Mg2+ respectively. The extrapolated Km for Mg2+ in the absence of the substrate is 0.73 mM. Imidodiphosphate-Mg2+ and free Ca2+ were used as competitive inhibitors of substrate and activator respectively. The equilibrium binding kinetics suggest an ordered mechanism for the activator and the substrate: Mg2+ ions bind the enzyme before PP(i)-Mg2+ in the formation of the catalytic complex, membrane-bound pyrophosphatase-(Mg2+)-(PP(i)-Mg2+).
Reaction centers with both light harvesting complexes I and II (B875 and B800/850; i.e., RCLHILHII complexes) have been isolated from Rhodobacter sphaeroides. These complexes have been incorporated into liposomes made from lipids purified from Escherichia coli. The electrochromic bandshift of carotenoids, present in these reconstituted complexes, shows shifted minima and maxima with respect to a similar spectrum in chromatophores of Rb. sphaeroides in a potassium diffusion potential induced difference spectrum (see also Crielaard, W., Hellingwerf, K.J. and Konings, W.N. (1989) Biochim. Biophys. Acta 973, 205-211). The absorbance spectrum, at room temperature or at 77 K, of both membrane preparations did not, however, reveal differences in the carotenoid region. The long-wavelength carotenoid peak in both preparations is located at 513 nm (77 K). A small difference could be observed between the 77 K excitation spectra of the B850 fluorescence. Reconstituted complexes show a carotenoid peak at 513 nm, whereas in chromatophores this peak is located at 514.5 nm. When fluorescence was recorded at 805 nm, to detect B800 excitation, there was a marked difference between both preparations. In liposomes the long wavelength B800-associated carotenoid peak is located at 512.5 nm, whereas in chromatophores this peak is located at 516 nm. These results explain the shifted minima and maxima in a potassium diffusion induced difference spectrum in proteoliposomes. The prediction of two carotenoid pools in chromatophores (De Grooth, B.G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 247-258) is confirmed, and the field sensitive carotenoids are identified as the pool that is associated with the B800 band (Kramer, H.J.M., Van Grondelle, R., Hunter, C.N., Westerhuis, W.H.J. and Amesz, J. (1984) Biochim. Biophys. Acta 765, 156-165).
The technique of distance measurement, utilizing spin relaxation enhancement by an external probe, has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion, Qi (also designated as Qn.- or Qc.-), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates. The location of the semiquinone anion is 6-10 A from the N side protein, establishing that there are two separate quinone reaction sites, i.e., 'Qi' and 'Qo', within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.