Hass Mathias A S, Vlasie Monica D, Ubbink Marcellus, Led Jens J
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Biochemistry. 2009 Jan 13;48(1):50-8. doi: 10.1021/bi801858f.
The dynamics of the reduced form of the blue copper protein pseudoazurin from Alcaligenes faecalis S-6 was investigated using (15)N relaxation measurements with a focus on the dynamics of the micro- to millisecond time scale. Different types of conformational exchange processes are observed in the protein on this time scale. At low pH, the protonation of the C-terminal copper-ligated histidine, His81, is observed. A comparison of the exchange rates in the presence and absence of added buffers shows that the protonation is the rate-limiting step at low buffer concentrations. This finding agrees with previous observations for other blue copper proteins, e.g., amicyanin and plastocyanin. However, in contrast to plastocyanin but similar to amicyanin, a second conformational exchange between different conformations of the protonated copper site is observed at low pH, most likely triggered by the protonation of His81. This process has been further characterized using CPMG dispersion methods and is found to occur with a rate of a few thousands per second. Finally, micro- to millisecond motions are observed in one of the loop regions and in the alpha-helical regions. These motions are unaffected by pH and are unrelated to the conformational changes in the active site of pseudoazurin.
利用（15）N弛豫测量研究了粪产碱菌S-6中蓝色铜蛋白假天青蛋白还原形式的动力学，重点关注微秒至毫秒时间尺度的动力学。在这个时间尺度上，在蛋白质中观察到了不同类型的构象交换过程。在低pH值下，观察到C端铜连接的组氨酸His81的质子化。添加缓冲液和不添加缓冲液时交换速率的比较表明，在低缓冲液浓度下，质子化是限速步骤。这一发现与之前对其他蓝色铜蛋白（如氨蓝蛋白和质体蓝素）的观察结果一致。然而，与质体蓝素不同但与氨蓝蛋白相似的是，在低pH值下，观察到质子化铜位点的不同构象之间发生了第二次构象交换，最有可能是由His81的质子化触发的。这个过程已经用CPMG色散方法进一步表征，发现其发生速率为每秒几千次。最后，在一个环区域和α螺旋区域观察到了微秒至毫秒的运动。这些运动不受pH值影响，与假天青蛋白活性位点的构象变化无关。
