Structure/function of the gamma regulatory dithiol domain of the chloroplast ATP synthase

Abstract

Strategically-placed dithiols act as photo-switches regulating the catalytic rates of several key enzymes in higher plants, thereby governing photosynthetic output. One such dithiol within the γ subunit of the chloroplast ATP synthase acts in concert with the inhibitory ϵ subunit to block nonproductive ATP hydrolysis, preventing depletion of essential ATP pools in the dark when photosynthetic electron transport is inactive. The critical γ dithiol is located within a special regulatory domain in the γ subunit of the F1 segment of the photosynthetic ATP synthase. We recently published a homology model of the three-dimensional structure of the γ subunit from higher plants that provides significant new insight into how the redox state of the dithiol may regulate catalysis. Studies described in this dissertation elucidate the mechanism by which this critical regulatory process occurs.

A series of deletion mutants were designed to structurally map the regulatory domain of the γ subunit as well as an additional extra loop region in the homology model identified as having a possible involvement in dithiol regulation. Deleting major regions of the regulatory domain, as well as the central portion of the extra loop, relieved the inhibition of ATPase activity normally observed after oxidation of the regulatory dithiol. Deletions within the regulatory domain did not affect inhibition of ATPase activity by the epsilon subunit, whereas deletions of the extra loop region severely impaired the ability of ϵ to inhibit ATPase activity. The data suggest a model by which the C-terminal arm of ϵ subunit binds across both the regulatory domain and the extra loop region of the γ subunit.

To determine the role of interdomain motion within the γ subunit, cysteines were engineered at locations expected to lead to cross-linking between the N- and C-terminal helices, and between the C-terminal helix and the central domain of the γ subunit. Cross-links formed in all cases but did not interfere with catalysis, suggesting certain large-scale domain movements are not necessary for dithiol regulation. The γV31C,A276C mutant reduced MgATPase activity and ATP synthesis, suggesting certain γ-β interactions are critical for proton coupled activation.

Single molecule fluorescence experiments were designed to characterize the dynamic motion of the regulatory domain by measuring changes in fluorescence amplitude of a thiol reactive probe placed on one of the two γ dithiols that form the regulatory disulfide. Autocorrelation time constants were generated from surface immobilized, labeled protein samples. Inhibiting the enzyme with AMP-PNP resulted in a significant increase in the frequency of fluorescence amplitude shifts indicating the presence of a nucleotide-dependent change in the conformation of the regulatory domain.