We have found that fumarase A can catalyze the transfer of O-18 from [2-O-18]malate and H-2 from (3R)[3-H-2]malate to the carbon skeleton of acetylenedicarboxylate to form O-18 and H-2 labeled enol oxalacetate. Our data indicates that 33% of the O-18 mobilized from [2-O-18]malate and close to 100% of the H-2 mobilized from (3R)-[3-H-2] malate would be transferred at an infinite concentration of acetylenedicarboxylate. This is the first report of oxygen transfer by an enzyme in the hydro-lyase class, but there have been previous reports of proton transfer by enzymes of this class. The transfer of the oxygen and proton removed by fumarase A in the dehydration of malate requires that these atoms remain associated with the enzyme long enough for the four carbon substrates to interchange in the active site. With certain assumptions, the results reported in this paper allow a rough calculation of the rate constants for the dissociation of the oxygen and proton from fumarase A. The rate constant for the oxygen is between 1 x 10(4) and 8 x 10(5) s(-1). Since fumarase A contains a [4Fe-4S] cluster that acts as a Lewis acid in the dehydration reaction catalyzed by fumarase A, it is likely that the oxygen is bound to the enzyme as a ligand to an iron atom in the cluster. The rate constant for the dissociation of oxygen from fumarase A compares favorably with the rate constants reported for water interchange on iron complexes. The rate constant for the dissociation of the proton from fumarase A is on the order of 7 x 10(4) s(-1). This is similar to the rate constant for the dissociation of a proton from a carboxylic acid but higher than the rate constants for the dissociation of protons from protonated amines, protonated imidazoles, thiols, and alcohols. If the base that removes the proton from C-3 of malate in the active site of fumarase A is not a carboxylate, the dissociation of the proton from this group must be accelerated in some way by the enzyme.