As a first step to determine the folding pathway of a
protein with an alpha/beta doubly
wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii
holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual
amide protons of holoflavodoxin were determined using the
hydrogen exchange method. The
amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the
amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the
protein. The results obtained are discussed with respect to their implications for
flavodoxin folding and for modulation of the
flavin redox potential by the
apoprotein. We do not find any evidence that A. vinelandii
holoflavodoxin II is divided into two subdomains based on its
amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly
wound protein Che Y.