A PAS Domain with an Oxygen Labile [4Fe-4S]2+ Cluster in the Oxygen Sensor Kinase NreB of Staphylococcus carnosus

• Published in: Biochemistry (Easton) 2008-12, Vol.47 (52), p.13921-13932
• Main Authors: Müllner, Martin, Hammel, Oliver, Mienert, Bernd, Schlag, Steffen, Bill, Eckhard, Unden, Gottfried
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi8014086

The cytoplasmic histidine sensor kinase NreB of Staphylococcus carnosus responds to O2 and controls together with the response regulator NreC the expression of genes of nitrate/nitrite respiration. nreBC homologous genes were found in Staphylococcus strains and Bacillus clausii, and a modified form was found in some Lactobacillus strains. NreB contains a sensory domain with similarity to heme B binding PAS domains. Anaerobically prepared NreB of S. carnosus exhibited a (diamagnetic) [4Fe-4S]2+ cluster when assessed by Mössbauer spectroscopy. Upon reaction with air, the cluster was degraded with a half-life of ∼2.5 min. No significant amounts of Mössbauer or EPR detectable intermediates were found during the decay, but magnetic Mössbauer spectra revealed formation of diamagnetic [2Fe-2S]2+ clusters. After extended exposure to air, NreB was devoid of a FeS cluster. Photoreduction with deazaflavin produced small amounts of [4Fe-4S]+, which were degraded subsequently. The magnetically perturbed Mössbauer spectrum of the [4Fe-4S]2+ cluster corroborated the S = 0 spin state and revealed uniform electric field gradient tensors of the iron sites, suggesting full delocalization of the valence electrons and binding of each of the Fe ions by four S ligands, including the ligand to the protein. Mutation of each of the four Cys residues inactivated NreB function in vivo in accordance with their role as ligands. [4Fe-4S]2+ cluster-containing NreB had high kinase activity. Exposure to air decreased the kinase activity and content of the [4Fe-4S]2+ cluster with similar half-lives. We conclude that the sensory domain of NreB represents a new type of PAS domain containing a [4Fe-4S]2+ cluster for sensing and function.