HYDROGEN-EXCHANGE IN THE CARBON-MONOXIDE COMPLEX OF SOYBEAN LEGHEMOGLOBIN

Hydrogen/deuterium exchange rates for individual amide protons have be en measured for the carbon monoxide complex of soybean leghemoglobin. Fast two-dimensional NOESY experiments were performed, with 5.2-min da ta-collection time for each spectrum, which made possible the measurem ent of NOE cross-peaks of relatively rapidly exchanging amide protons at early time points. Exchange rates were measured for 61 backbone ami des, and protection factors were calculated to provide information on the packing and local stability of the protein. The data are consisten t with the presence of transient cooperative local unfolding of helica l segments. The B-, E-, G and H-helices have extensive regions of slow -, medium- and fast-exchanging amide protons. For each of these helice s, there is a progressive decrease in protection on moving from the he lix center to the termini. This is consistent with a stable helix cent er, with dynamic fraying at the ends. Amide exchange from the A-helix and C-helix is rapid except in small local regions. The F-helix, which is located on the proximal side of the heme pocket and is well formed in solution as demonstrated by characteristic medium range NOE connec tivities [Morikis, D., Lepre, C. A. & Wright, P. E. (1994) Eur. J. Bio chem. 219, 611-626], exhibits fast exchange for all amide protons. The implied flexibility and low stability of the F-helix may be functiona lly important in facilitating movement of the helix upon ligand bindin g. Fast exchange has also been observed far all amide protons in the C E-loop and in turns, as expected for flexible or solvent exposed regio ns. A strong tertiary contact has been established between the A-, G- and H-helices by the presence of a slowly exchanging indole NepsilonH of Trp129.