The M r of spinach chloroplast coupling factor 1 has been determined by sedimentation equilibrium and by light scattering to be 400 000 ± 24 600 and 407 000 ± 20 000, respectively. These values differ substantially from that obtained previously (325 000) and are consistent with an α3β3γδϵ subunit stoichiometry.
Fluorescent probes were attached to the single sulfhydryl residue on the isolated epsilon polypeptide of chloroplast coupling factor 1 (CF1), and the modified polypeptide was reconstituted with the epsilon-deficient enzyme. A binding stoichiometry of one epsilon polypeptide per CF1 was obtained. This stoichiometry corresponded to a maximum inhibition of the Ca2+-dependent ATPase activity of the enzyme induced by epsilon removal. Resonance energy transfer between the modified epsilon polypeptide and fluorescent probes attached to various other sites on the enzyme allowed distance measurements between these sites and the epsilon polypeptide. The epsilon-sulfhydryl is nearly equidistant from both the disulfide (23 A) and the dark-accessible sulfhydryl (26 A) of the gamma subunit. Measurement of the distance between epsilon and the light-accessible gamma-sulfhydryl was not possible due to an apparent exclusion of modified epsilon from epsilon-deficient enzyme after modification of the light-accessible site. The distances measured between epsilon and the nucleotide binding sites on the enzyme were 62, 66, and 49 A for sites 1, 2, and 3, respectively. These measurements place the epsilon subunit in close physical proximity to the sulfhydryl-containing domains of the gamma subunit and approximately 40 A from the membrane surface. Enzyme activity measurements also indicated a close association between the epsilon and gamma subunits: epsilon removal caused a marked increase in accessibility of the gamma-disulfide bond to thiol reagents and exposed a trypsin-sensitive site on the gamma subunit. Either disulfide bond reduction or trypsin cleavage of gamma significantly enhanced the Ca2+-ATPase activity of the epsilon-deficient enzyme. Thus, the epsilon and gamma polypeptides of coupling factor 1 are closely linked, both physically and functionally.
The gene encoding the E subunit (atpf?) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia COK The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol t o obtain E that is as biologically active as E purified from chloroplastcoupling factor 1 (CF,). Recombinant E folded i n this manner inhibits the ATPase activity of soluble and membrane-bound CF, deficient in e and restores proton impermeability to thylakoid membranes reconstituted with CF, deficient i n E . Site-directed mutagenesis was used to generate truncations and single amino acid substitutions i n the primary structure of E. I n the five mutants tested, alterations that weaken ATPase inhibition by recombinant E affect its ability t o restore proton impermeability t o a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeabilty. As in the case of E. coli, it appears that N-terminal truncations of the 4 subunit have more profound effects than C-terminal deletions on the function of E. Recombinant E with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the E of spinach and the E of Pisum sativum, inhibits ATPase activity with a reduced potency similar t o that of purified pea E . Four of the six amino acids are serine or threonine.These hydroxylated amino acids may be important in E-CF, interactions.The chloroplast ATP synthase (CF,CFo) catalyzes the phosphorylation of ADP to ATP at the expense of the proton gradient. The ATP synthase is biochemically separable into two parts: CF,, a membrane-integrated protein complex that translocates protons, and CF,, the catalytic portion of the CF,CFo complex. CF, consists of five different types of subunits, denoted a, p, y, 6, and E, in order of decreasing molecular weight, with a stoichiometry of a,P,y8~. On illuminated thylakoid membranes, CF, catalyzes ATP synthesis at high rates. In the dark, the enzyme usually does not catalyze ATP hydrolysis, even though hydrolysis is thermodynamically favorable. Thus, when thylakoid membranes are illuminated, the ATP synthase activity is rapidly switched on (McCarty et al., 1988).The transition of CF,CFo from an active state to an inactive state is tightly regulated. The suppression of wasteful ATPase activity in the dark requires the presence Supported by National Science Foundation grants MCB 94
lageindorf and Evanis (15) reported that the rates of 2, 3', 6-trichlorophenolindophenol (TCPIP)5 reduction by chloroplasts isolated from the primary leaves of red kidney bean are strongly dependent uI)on the pH of the buffer in which the leaves are homogenized. Grindiing leaves at pH values below 8 resulted in chloroplasts whose Hill reaction was rapidly and irreversibly inhibited. In contrast, the chloroplasts of sonie plants, including those from spinach, are not inactivated under these conditions. Clendenning and Gorham (7) Chloroplasts were prepared in a variety of grinding media as described previously (14) and were usually used unwashed. Chlorophyll was determined by the method of Arnon (2).
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