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Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca 2+ for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn 2+ EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn 2+ with a K D of 0.05 to 0.35 μM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five β-sheet helices within the repeat domain of the toxin upon binding Ca 2+ is required for cell intoxication.