Cytochrome c Recognition of Immobilized, Orientational Variants of Cytochrome b₅: Direct Force and Equilibrium Binding Measurements

Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b 5 (cyt b 5 ) on its interactions with cytochrome c (cyt c ). In this work, we used two cyt b 5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b 5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b 5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b 5 . At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.