Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, involved in neuroprotection. O2 equilibrium measurements on mouse Ngb yielded significantly different P50 values, ranging from 2 torr to 10 torr. By a kinetic approach minimizing the effects of protein autoxidation, we measured P50 = 2.2 torr at 20 C. As predicted from the structure, O2 binds to the Y44D Ngb mutant more quickly (k = 2.2 s1 vs 0.15 s1) and with slightly higher affinity (P50 = 1.3 torr) than wild-type. In addition, we introduced a novel reduction protocol for metNgb based on NADH:flavorubredoxin oxidoreductase (FlRd-red) from Escherichia coli, a candidate for the Ngb reducing activity recently identified in E. coli extracts. Interestingly, E. coli FlRd-red shares sequence similarity with the FAD-binding domain of the human apoptosis-inducing factor, a finding which may have unexpected significance with reference to the mechanism of neuroprotection by Ngb.
Neuroglobin: enzymatic reduction and oxygen affinity.
2008
Abstract
Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, involved in neuroprotection. O2 equilibrium measurements on mouse Ngb yielded significantly different P50 values, ranging from 2 torr to 10 torr. By a kinetic approach minimizing the effects of protein autoxidation, we measured P50 = 2.2 torr at 20 C. As predicted from the structure, O2 binds to the Y44D Ngb mutant more quickly (k = 2.2 s1 vs 0.15 s1) and with slightly higher affinity (P50 = 1.3 torr) than wild-type. In addition, we introduced a novel reduction protocol for metNgb based on NADH:flavorubredoxin oxidoreductase (FlRd-red) from Escherichia coli, a candidate for the Ngb reducing activity recently identified in E. coli extracts. Interestingly, E. coli FlRd-red shares sequence similarity with the FAD-binding domain of the human apoptosis-inducing factor, a finding which may have unexpected significance with reference to the mechanism of neuroprotection by Ngb.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
