Interaction between Cytochrome caa3 and F1F0-ATP Synthase of Alkaliphilic Bacillus pseudofirmus OF4 Is Demonstrated by Saturation Transfer Electron Paramagnetic Resonance and Differential Scanning Calorimetry Assays

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Abstract
Interaction between the cytochrome caa3 respiratory chain complex and F1F0-ATP synthase from extremely alkaliphilic Bacillus pseudofirmus OF4 has been hypothesized to be required for robust ATP synthesis by this alkaliphile under conditions of very low protonmotive force. Here, such an interaction was probed by differential scanning calorimetry (DSC) and by saturation transfer electron paramagnetic resonance (STEPR). When the two purified complexes were embedded in phospholipid vesicles individually [(caa3)PL, (F1F0)PL)] or in combination [(caa3 + F1F0)PL] and subjected to DSC analysis, they underwent exothermic thermodenaturation with transition temperatures at 69, 57, and 46/75 °C, respectively. The enthalpy change, ΔH (−8.8 kcal/mmol), of protein−phospholipid vesicles containing both cytochrome caa3 and F1F0 was smaller than that (−12.4 kcal/mmol) of a mixture of protein−phospholipid vesicles formed from each individual electron transfer complex [(caa3)PL + (F1F0)PL]. The rotational correlation time of spin-labeled caa3 (65 μs) in STEPR studies increased significantly when the complex was mixed with F1F0 prior to being embedded in phospholipid vesicles (270 μs). When the complexes were reconstituted separately and then mixed together, or either mitochondrial cytochrome bc1 or F1F0 was substituted for the alkaliphile F1F0, the correlation time was unchanged (65−70 μs). Varying the ratio of the two alkaliphile complexes in both the DSC and STEPR experiments indicated that the optimal stoichiometry is 1:1. These results demonstrate a physical interaction between the cytochrome caa3 and F1F0-ATP synthase from B. pseudofirmus OF4 in a reconstituted system. They support the suggestion that such an interaction between these complexes may contribute to sequestered proton transfers during alkaliphile oxidative phosphorylation at high pH.

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