Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin

Tentoxin, produced by phytopathogenic fun,oi, selectively affects the funct ion of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K-i approximate to 10 nM) site on the chlo roplast F-1 (CF1) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K-d > 10 muM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown t o be due, in part,. to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF1. An aspartate in this position is requ ired, but is not sufficient, for tentoxin inhibition. By comparison with th e solved structure of mitochondrial F-1 [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probab ly located at an interface between alpha and beta subunits on CF1 where res idues on the alpha subunit could also participate in tentoxin binding. A hy brid core F-1 enzyme assembled with beta and gamma subunits of the tentoxin -sensitive spinach CF1, and an alpha subunit of the tentoxin-insensitive ph otosynthetic bacterium Rhodospirillum rubrum F-1 (RrF(1)), was stimulated b ut not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem, 268, 2179-2186]. In this study, chi meric al subunits were prepared by introducing short segments of the spinac h CF1 alpha sub-unit from a poorly conserved region which is immediately ad jacent to beta -Asp83 in the crystal structure, into equivalent positions i n the RrF(1) a subunit using oligonucleotide-directed mutagenesis. Hybrid e nzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. C hanging beta -Asp83 to leucine resulted in loss of both inhibition and stim ulation by tentoxin in the chimeras. The results indicate that tentoxin inh ibition requires additional alpha residues that are not present on the RrF( 1) alpha subunit, A structural model of a putative inhibitory tentoxin bind ing pocket is presented.