O 2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form

Abstract

FixL, a rhizobial heme-based O 2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O 2 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.