The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme (EFE) from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coil, were purified to homogeneity, and assayed in vitro for K-m, k(cat) and thermostability. The relative k(cat) of two mutated EFEs, H305Q and H335Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The k(cat) values for other mutant enzymes were 11- to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs (but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30 degrees C were 11- to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the k(cat) value (relative k(cat) was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the purified mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.