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Properties of bound inorganic phosphate on bovine mitochondrial F1F0-ATP synthase
1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Abstract:
Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi <--> ATP exchange reaction with bound Pi. The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi <--> ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers.
Insights
Dimethyl sulfoxide (DMSO) treatment of beef-heart mitochondrial F1F0-ATP synthase reveals inorganic phosphate (Pi) binding sites. DMSO facilitates Pi exchange, uncovering catalytic and noncatalytic binding sites involved in ATP synthesis.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial Function
Background:
- Beef-heart mitochondrial F1F0-ATP synthase is crucial for cellular energy production.
- Understanding inorganic phosphate (Pi) binding is key to elucidating ATP synthesis mechanisms.
- Previous studies indicated multiple Pi binding sites on ATP synthase.
Purpose of the Study:
- To investigate the binding and exchange dynamics of inorganic phosphate (Pi) on F1F0-ATP synthase.
- To identify the roles of different Pi binding sites, including catalytic and noncatalytic sites.
- To explore the effect of dimethyl sulfoxide (DMSO) on Pi binding and ATP synthesis.
Main Methods:
- Enzyme treatment with dimethyl sulfoxide (DMSO) and subsequent buffer exchange.
- Incubation with radiolabeled inorganic phosphate (32Pi) and adenosine triphosphate (ATP).
- Analysis of Pi and ATP binding and exchange using pyrophosphate and chemical modification.
Main Results:
- DMSO treatment enabled complete exchange of six bound inorganic phosphate (Pi) molecules with 32Pi.
- Two Pi molecules bound at a noncatalytic site were resistant to ATP displacement but removed by pyrophosphate.
- Three Pi molecules at catalytic sites participated in ATP synthesis and Pi-ATP exchange, consistent with crystallographic data.
Conclusions:
- F1F0-ATP synthase possesses at least five functionally distinct inorganic phosphate (Pi) binding sites.
- DMSO is a valuable tool for probing Pi binding sites and dynamics in ATP synthase.
- The study clarifies the roles of catalytic and noncatalytic Pi binding sites in energy transduction.