Müllner Martin, Hammel Oliver, Mienert Bernd, Schlag Steffen, Bill Eckhard, Unden Gottfried
Institut fur Mikrobiologie and Weinforschung, Universitat Mainz, Becherweg 15, 55099 Mainz, Germany.
Biochemistry. 2008 Dec 30;47(52):13921-32. doi: 10.1021/bi8014086.
The cytoplasmic histidine sensor kinase NreB of Staphylococcus carnosus responds to O(2) 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 cluster when assessed by Mossbauer spectroscopy. Upon reaction with air, the cluster was degraded with a half-life of approximately 2.5 min. No significant amounts of Mossbauer or EPR detectable intermediates were found during the decay, but magnetic Mossbauer spectra revealed formation of diamagnetic 2Fe-2S 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 Mossbauer spectrum of the 4Fe-4S 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 cluster-containing NreB had high kinase activity. Exposure to air decreased the kinase activity and content of the 4Fe-4S cluster with similar half-lives. We conclude that the sensory domain of NreB represents a new type of PAS domain containing a 4Fe-4S cluster for sensing and function.
肉葡萄球菌的细胞质组氨酸传感激酶NreB对氧气作出反应，并与反应调节因子NreC共同控制硝酸盐/亚硝酸盐呼吸相关基因的表达。在葡萄球菌菌株和克劳氏芽孢杆菌中发现了nreBC同源基因，在一些乳酸杆菌菌株中发现了其修饰形式。NreB包含一个与血红素B结合PAS结构域相似的传感结构域。通过穆斯堡尔光谱评估，厌氧制备的肉葡萄球菌NreB呈现出（抗磁性的）4Fe-4S簇。与空气反应后，该簇以约2.5分钟的半衰期降解。在衰变过程中未发现大量穆斯堡尔或电子顺磁共振可检测的中间体，但磁性穆斯堡尔光谱显示形成了抗磁性的2Fe-2S簇。长时间暴露于空气中后，NreB不含FeS簇。用脱氮黄素进行光还原产生少量的4Fe-4S，随后它们被降解。4Fe-4S簇的磁性扰动穆斯堡尔光谱证实了S = 0自旋态，并揭示了铁位点均匀的电场梯度张量，表明价电子完全离域，每个铁离子由四个硫配体结合，包括与蛋白质的配体。四个半胱氨酸残基中的每一个发生突变都会使NreB在体内的功能失活，这与其作为配体的作用一致。含有4Fe-4S簇的NreB具有高激酶活性。暴露于空气中会降低激酶活性和4Fe-4S簇含量，且半衰期相似。我们得出结论，NreB的传感结构域代表了一种新型的PAS结构域，其包含用于传感和功能的4Fe-4S簇。
