Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F₀F₁‐ATPase

Abstract

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the V max . There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit : is differently exposed to trypsin.

Energy-dependent Transformation of F0·F1-ATPase in Paracoccus denitrificans Plasma Membranes

“…This explanation is in accord with the previously reported data of Harris et al (30), who have shown that P. denitrificans coupling ATPase contains tightly bound nucleotides, which became exchangeable upon the membrane energization. Our data somehow agree with those recently reported by PachecoMoisés et al (34). However, their speculative proposal on two conformationally different active states of the enzyme as induced by either succinate or sulfite can hardly be deduced from the activating effect of trypsin on the ATPase activity in such a complex system as vesicular preparation of the bacterial membranes.…”

“…In some respects, F 0 ⅐F 1 synthase of P. denitrificans seemed to be remarkably similar to the mitochondrial ADP(Mg 2ϩ )-inhibited enzyme form stabilized by azide (22) or to the thermophilic Bacillus PS3 mutant F 0 ⅐F 1 , which is incapable of ATP binding to the "non-catalytic" sites on ␣-subunits (33). This similarity is corroborated by recent reports demonstrating that the ATPase activity of coupled vesicles derived from P. denitrificans is susceptible to activation by sulfite and/or by energization (34,35). Analogous phenomena have been reported for chloroplasts (36), photosynthetic purple bacteria (37), cyanobacterium Synechococcus 6716 (38,39), E. coli (40), and the mitochondrial enzyme (41).…”

“…It has been shown that sulfite prevents and reverses the ADP-induced inhibition of the mitochondrial (9) and chloroplast (36) F 1 -ATPases. Sulfite has also been shown to stimulate bacterial F 1 -ATPases, particularly that of P. denitrificans (34). It seemed thus logical to check whether sulfite is able to protect the ⌬ H ϩ-activated ATPase against further deactivation upon de-energization.…”

Purification and Biochemical Characterization of the F1-ATPase fromAcidithiobacillus ferrooxidansNASF-1 and Analysis of theatpOperon

“…28) In Paracoccus denitrificans, sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased the V max . 29) Since sulfite is an intermediate of sulfur oxidation in A. ferrooxidans, activation of ATPase activity by sulfite ion appeared to be possible in A. ferrooxidans cell.…”

Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase fromParacoccus denitrificansat 2.3 Å resolution

“…Other eubacterial F-ATPases, exemplified by those from C. thermarum (Cook et al, 2003) and P. denitrificans (Zharova & Vinogradov, 2012), can synthesize ATP in the presence of a proton motive force, but their ATP hydrolase activity is inhibited in its absence (Pacheco-Moisé s et al, 2000. The mechanism of inhibition in C. thermarum is not understood, but in P. denitrificans and other -proteobacteria the inhibition of ATP hydrolysis involves an inhibitor protein known as the inhibitor protein (Morales-Ríos et al, 2010).…”