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首页> 外文期刊>Biochemistry >Breaking the Proximal Fe~(II)-N_(His) Bond in Heme Proteins through Local Structural Tension: Lessons from the Heme b Proteins Nitrophorin 4, Nitrophorin 7, and Related Site-Directed Mutant Proteins
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Breaking the Proximal Fe~(II)-N_(His) Bond in Heme Proteins through Local Structural Tension: Lessons from the Heme b Proteins Nitrophorin 4, Nitrophorin 7, and Related Site-Directed Mutant Proteins

机译：通过局部结构张力打破血红素蛋白中近端的Fe〜（II）-N_（His）键：血红素b蛋白亚硝酸盐4，亚硝酸盐7和相关的定点突变蛋白的教训

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The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NPI-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues G1u27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe~(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe~(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe~(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe~(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itSel.f. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe~(II)-His bond breakage in heme proteins.
机译：导致铁血红蛋白可溶性鸟苷酸环化酶（sGC）中近端铁-组氨酸键断裂的因素仍是一个争论的问题。此事件是导致NO产生第二信使分子cGMP的关键机制。出乎意料的是，在血红素蛋白氮荧光蛋白7（NP7）中，我们通过UV-vis吸收光谱和共振拉曼光谱发现，血红素的减少导致近端组氨酸配体的丧失，而对于其他异蛋白（NPI-4）则没有观察到。 。结构上的考虑导致定点突变体NP7（E27V），NP7（E27Q），NP4（D70A）和NP2（V24E）的产生和光谱研究。这些蛋白质的光谱研究表明，NP7近端血红素袋中残基G1u27，Phe43和His60的空间排列是通过空间需求削弱Fe〜（II）-His60键的原因。对用2，4-二甲基氘杂亚血红素（“对称血红素”）重构的NP7样品的光谱研究表明，血红素乙烯基取代基也是原因。尽管在未配位的铁血红蛋白中很少见到铁-组氨酸键的断裂，但是由于NO的反式反作用，有时会观察到NO与第六个配位点结合时Fe〜（II）-His键的断裂。但是，在血红素蛋白中仍然很少见，这与反式配体亚硝酰基模型血红素的情况相反。因此，出现了由哪些因素决定蛋白质中Fe〜（II）-His键的不饱和化的问题。令人惊讶的是，突变体NP2（V24E）的行为与sGC特别相似。即，Fe〜（II）-His键对NO结合时的断裂敏感，而未配体形式则在中性pH下结合近端His。据我们所知，NP2（V24E）是第一个实例，其中通过His-on His-off开关的功能通过近端His itSel.f以外的定点诱变工程化为血红素蛋白。因此，引入立体张力作为血红素蛋白近端Fe〜（II）-His键断裂的潜在结构决定因素。

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