Pinakoulaki Eftychia, Ohta Takehiro, Soulimane Tewfik, Kitagawa Teizo, Varotsis Constantinos
Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece.
J Am Chem Soc. 2005 Nov 2;127(43):15161-7. doi: 10.1021/ja0539490.
Reaction pathways in the enzymatic formation and cleavage of the N-N and N-O bonds, respectively, are difficult to verify without the structure of the intermediates, but we now have such information on the heme a(3)(2+)-NO species formed in the reaction of ba(3)-oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a(3)(2+)-NO/Cu(B)(1+) species by its characteristic Fe-NO and N-O stretching frequencies at 539 and 1620 cm(-)(1), respectively. The Fe-NO and N-O frequencies in ba(3)-oxidase are 21 and 7 cm(-)(1) lower and higher, respectively, than those observed in Mb-NO. From these results and earlier Raman and FTIR measurements, we demonstrate that the protein environment of the proximal His384 that is part of the Q-proton pathway controls the strength of the Fe-His384 bond upon ligand (CO vs NO) binding. We also show by time-resolved FTIR spectroscopy that Cu(B)(1+) has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N(2)O by ba(3)-oxidase proceeds by the fast binding of the first NO molecule to heme a(3) with high-affinity, and the second NO molecule binds to Cu(B) with low-affinity, producing the temporal co-presence of two NO molecules in the heme-copper center. The low-affinity of Cu(B) for NO binding also explains the NO reductase activity of the ba(3)-oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a(3)(2+)-NO/Cu(B)(1+) species, the structure of the binuclear heme a(3)-Cu(B)(1+) center in the initial step of the NO reduction mechanism is known.
在没有中间体结构的情况下，分别用于酶促形成和断裂N-N键与N-O键的反应途径很难得到验证，但我们现在通过共振拉曼光谱得到了关于ba(3)-氧化酶与NO反应形成的血红素a(3)(2+)-NO物种的此类信息。我们已通过其分别在539和1620 cm(-)(1)处的特征性Fe-NO和N-O伸缩频率鉴定出His-血红素a(3)(2+)-NO/Cu(B)(1+)物种。ba(3)-氧化酶中的Fe-NO和N-O频率分别比在Mb-NO中观察到的频率低21 cm(-)(1)和高7 cm(-)(1)。根据这些结果以及早期的拉曼和傅里叶变换红外光谱测量，我们证明作为Q-质子途径一部分的近端His384的蛋白质环境在配体（CO与NO）结合时控制着Fe-His384键的强度。我们还通过时间分辨傅里叶变换红外光谱表明，Cu(B)(1+)对NO的亲和力比对CO的亲和力低得多。我们认为ba(3)-氧化酶将NO还原为N(2)O是通过第一个NO分子以高亲和力快速结合到血红素a(3)上进行的，而第二个NO分子以低亲和力结合到Cu(B)上，从而在血红素-铜中心产生两个NO分子的瞬时共存。Cu(B)对NO结合的低亲和力也解释了ba(3)-氧化酶与其他血红素-铜氧化酶不同的NO还原酶活性。随着His-血红素a(3)(2+)-NO/Cu(B)(1+)物种的鉴定，NO还原机制初始步骤中双核血红素a(3)-Cu(B)(1+)中心的结构已为人所知。
