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Transduction of electrochemical proton gradient into ATP synthesis is performed by F 1 F O -ATP synthase. The reverse reaction is prevented by the regulatory subunit Inh1. Knockout of the inh1 gene in the basidiomycete Ustilago maydis was generated in order to study the function of this protein in the mitochondrial metabolism and cristae architecture. Deletion of inh1 gen did not affect cell growth, glucose consumption, and biomass production. Ultrastructure and fluorescence analyzes showed that size, cristae shape, network, and distribution of mitochondria was similar to wild strain. Membrane potential, ATP synthesis, and oxygen consumption in wild type and mutant strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of V max and K M for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated V max values 4-times higher for the mutant strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed. ATPase activity of WT oligomers was stimulated several times by dodecyl-maltoside (DDM), probably by removal of ADP from F 1 sector, while DDM induced an inactive form of the mutant oligomers. • Inh1 gen deletion did not modify the mitochondrial cristae architecture, either cells growth, or ATP synthesis. • Inh1 gen deletion did not affect the dimeric state of complex V. • Dimer from inh1 Δ is 7-times more active than dimer from WT. • Dodecyl-maltoside (DDM) relieves ADP-inhibition of dimer and monomer of WT strain, increasing the ATPase activity. • ATPase activity of the dimer and monomer from inh1 Δ was sensitive to DDM presence. [ABSTRACT FROM AUTHOR]