FixL, a rhizobial heme-based O-2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low 02 tension, while the kinase activity is inhibited in the case of the O-2-bound form. The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported. Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations. The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage. On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O-2 from CO and NO, and signals the O-2-bound state by downregulating the phosphoryl transfer reaction. (C) 2003 Elsevier Science (USA). All rights reserved.