Clark Robert W, Youn Hwan, Parks Ryan B, Cherney Melisa M, Roberts Gary P, Burstyn Judith N
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biochemistry. 2004 Nov 9;43(44):14149-60. doi: 10.1021/bi0487948.
A unique feature of CooA, a heme-containing transcription factor, is that the N-terminal proline is the distal heme ligand in the ferrous state, and this ligand is displaced upon CO binding. To investigate the importance of Pro(2) in CO-dependent DNA binding, several CooA variants that alter N-terminal ligation were characterized. Electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectra of these variants provide the most definitive evidence that Pro(2) is the distal ligand in Fe(III) CooA. Furthermore, the functional and spectroscopic properties of these proteins depended on whether a weak ligand occupied the distal heme coordination site: for CooA variants in which distal coordination is disrupted, the DNA-binding affinities and Fe(II)-CO spectral properties showed an unexpected dependence on the order of CO addition and heme reduction. If N-terminal variant samples were incubated with CO before the heme was reduced, the proteins displayed DNA-binding affinities and Fe(II)-CO spectral characteristics similar to those of wild-type (WT) CooA. However, if the same samples were incubated with CO after the heme was reduced, the extent of functional and spectral similarity to WT CooA negatively correlated with the amount of high-spin heme present in the ferric state. From these data, it was inferred that the absence of a distal heme ligand in the ferric state prevents WT-like CO binding to the ferrous state, and it was hypothesized that correct CO binding is inhibited by the collapse of the distal heme pocket upon reduction. Together with the observation that L116H CooA, a variant in which His(116) replaces Pro(2) as the distal heme ligand, binds CO more slowly than WT CooA, these data indicate that the presence of a weak distal heme ligand, not specifically ligation by the N-terminal proline, is crucial for proper function. The role of Pro(2) in CooA is apparently to direct CO to bind on the distal side of heme and to help maintain the integrity of the distal heme pocket during the redox-mediated ligand switch.
含血红素的转录因子CooA的一个独特特征是，N端脯氨酸在亚铁状态下是血红素的远端配体，并且该配体在CO结合时会被取代。为了研究Pro(2)在CO依赖性DNA结合中的重要性，对几个改变N端连接的CooA变体进行了表征。这些变体的电子吸收光谱、电子顺磁共振光谱和磁圆二色光谱提供了最确凿的证据，证明Pro(2)是Fe(III) CooA中的远端配体。此外，这些蛋白质的功能和光谱性质取决于远端血红素配位位点是否被一个弱配体占据：对于远端配位被破坏的CooA变体，DNA结合亲和力和Fe(II)-CO光谱性质显示出对CO添加顺序和血红素还原的意外依赖性。如果在血红素还原之前将N端变体样品与CO孵育，这些蛋白质表现出与野生型（WT）CooA相似的DNA结合亲和力和Fe(II)-CO光谱特征。然而，如果在血红素还原之后将相同的样品与CO孵育，与WT CooA的功能和光谱相似程度与三价铁状态下高自旋血红素的含量呈负相关。从这些数据可以推断，三价铁状态下远端血红素配体的缺失会阻止WT样的CO与亚铁状态结合，并且推测正确的CO结合会因还原时远端血红素口袋的塌陷而受到抑制。连同His(116)取代Pro(2)作为远端血红素配体的变体L116H CooA比WT CooA结合CO更慢的观察结果，这些数据表明存在一个弱的远端血红素配体，而不是N端脯氨酸的特异性连接，对于正常功能至关重要。Pro(2)在CooA中的作用显然是引导CO在血红素的远端结合，并在氧化还原介导的配体转换过程中帮助维持远端血红素口袋的完整性。
