Comparison between single-molecule and X-ray crystallography data on yeast F(1)-ATPase

Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F(1)-ATPase, mostly based on F(1) from thermophilic bacteria. In contrast, high-resolution crystal structures are only available for mitochondrial F(1). Here we present high resolution single molecule rotational data on F(1) from Saccharomyces cerevisiae, obtained using new high throughput detection and analysis tools. Rotational data are presented for the wild type mitochondrial enzyme, a “liver” isoform, and six mutant forms of yeast F(1) that have previously been demonstrated to be less efficient or partially uncoupled. The wild-type and “liver” isoforms show the same qualitative features as F(1) from Escherichia coli and thermophilic bacteria. The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in V(max), with magnitudes consistent with the level of disruption seen in the crystal structures. At least one of the mutant forms shows a previously un-observed dwell at the ATP binding angle, potentially attributable to slowed release of ADP. We discuss the correlation between crystal structures and single molecule results.