Budker V, Du J L, Seiter M, Eaton G R, Eaton S S
Department of Chemsitry, University of Denver, Colorado 80208, USA.
Biophys J. 1995 Jun;68(6):2531-42. doi: 10.1016/S0006-3495(95)80436-4.
Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.
通过双脉冲电子自旋回波光谱法和饱和恢复电子顺磁共振（EPR），在6至120 K之间测量了自旋标记的低自旋高铁血红蛋白的硝酰自由基电子自旋弛豫时间。通过饱和恢复在8至25 K之间以及通过电子自旋回波在4.2至20 K之间测量了氰高铁血红蛋白和咪唑高铁血红蛋白的自旋晶格弛豫时间。在低温下，铁电子自旋弛豫速率相对于铁-硝酰电子-电子自旋-自旋分裂较慢。随着温度升高，Fe(III)的弛豫速率变得与自旋-自旋分裂相当，然后大于自旋-自旋分裂，这使连续波EPR光谱中的分裂消失，并导致硝酰电子自旋回波衰减速率先增加后降低。在所研究的整个温度范围内，与Fe(III)的相互作用增加了硝酰的自旋晶格弛豫速率(1/T1)。所测量的Fe(III)的弛豫时间用于分析相互作用的硝酰的自旋回波衰减和饱和恢复(T1)数据中随温度变化的情况，并确定自旋间距离r。三种自旋标记的高铁血红蛋白的r值在15至15.5 Å之间，通过电子自旋回波和饱和恢复获得的值之间具有良好的一致性。对硝酰自旋回波和饱和恢复数据的分析还提供了在铁弛豫速率太快而无法通过饱和恢复或电子自旋回波光谱法直接测量的温度下的铁弛豫速率值。这些结果证明了使用时域EPR测量来探测蛋白质中缓慢弛豫的自旋与相对快速弛豫的金属之间距离的能力。
