APPARENT LOCAL STABILITY OF THE SECONDARY STRUCTURE OF AZOTOBACTER-VINELANDII HOLOFLAVODOXIN-II AS PROBED BY HYDROGEN-EXCHANGE - IMPLICATIONS FOR REDOX POTENTIAL REGULATION AND FLAVODOXIN FOLDING

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the H-1, C-13, and N-15 backbone che mical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues ) have been determined using multidimensional NMR spectroscopy. Its se condary structure is shown to contain a five-stranded parallel P-sheet (beta 2-beta 1-beta 3-beta 4-beta 5) and five alpha-helices, Exchange rates for the individual amide protons of haloflavodoxin were determi ned using the hydrogen exchange method. The amide protons of 65 residu es distributed throughout the structure of holoflavodoxin exchange slo wly at pH 6.2 (k(ex) < 10(-5) s(-1)) and can be used as probes in fut ure 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 th e apo state of the protein. The results obtained are discussed with re spect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any ev idence that A. vinelandii holoflavodoxin II is divided into two subdom ains based on its amide proton exchange rates, as opposed to what is f ound for the structurally but not sequentially homologous alpha/beta d oubly wound protein Che Y.