Department of Chemistry, University of Cyprus, Nicosia, Cyprus.
J Phys Chem B. 2011 Nov 10;115(44):13012-8. doi: 10.1021/jp205944e. Epub 2011 Oct 13.
Fourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.5 range, suggesting the presence of two conformations at the active site. The observed C-O frequencies are 5 and 16 cm(-1) lower and higher, respectively, than that obtained previously (Oinuma, K.-I.; et al. FEBS Lett.2004, 568, 44-48). We suggest that the strength of the Fe-His bond and the neutralization of the negatively charged propionate groups modulate the ν(Fe-CO)/ν(CO) back-bonding correlation. The "light" minus "dark" difference FTIR spectra indicate that the heme propionates are in both the protonated and deprotonated forms, and the photolyzed CO becomes trapped within a ligand docking site (ν(CO) = 2138 cm(-1)). The TRS(2)-FTIR spectra show that the rate of recombination of CO to the heme is k(1945 cm(-1)) = 126 ± 20 s(-1) and k(1964 cm(-1)) = 122 ± 20 s(-1) at pH 5.6, and k(1945 cm(-1)) = 148 ± 30 s(-1) and k(1964 cm(-1)) = 158 ± 32 s(-1) at pH 8.5. The rate of decay of the heme propionate vibrations is on a time scale coincident with the rate of rebinding, suggesting that there is a coupling between ligation dynamics in the distal heme environment and the environment sensed by the heme propionates. The implications of these results with respect to the proximal His-Fe heme environment including the propionates and the positively charged or proton-donating residues in the distal pocket which are crucial for the synthesis of nitriles are discussed.
傅里叶变换红外（FTIR）光谱、“光”减去“暗”差 FTIR 光谱和时间分辨分步扫描（TRS(2)）FTIR 光谱报道了一氧化碳醛肟脱水酶。已经鉴定出位于 1945 和 1964 cm(-1)处的血红素的两个 C-O 模式，并且在 H(2)O/D(2)O 交换和 pH 5.6-8.5 范围内保持不变，表明活性部位存在两种构象。观察到的 C-O 频率分别比以前（Oinuma，K.-I.；等人，FEBS Lett.2004，568，44-48）获得的频率低 5 和 16 cm(-1)。我们认为，Fe-His 键的强度和带负电荷的丙酸盐基团的中和调节了 ν(Fe-CO)/ν(CO)的反向键合相关性。“光”减去“暗”差 FTIR 光谱表明血红素丙酸盐处于质子化和去质子化形式，光解 CO 被困在配体 docking 位点内（ν(CO) = 2138 cm(-1)）。TRS(2)-FTIR 光谱表明，在 pH 5.6 时，CO 与血红素的重组速率为 k(1945 cm(-1)) = 126 ± 20 s(-1)和 k(1964 cm(-1)) = 122 ± 20 s(-1)，而在 pH 8.5 时，k(1945 cm(-1)) = 148 ± 30 s(-1)和 k(1964 cm(-1)) = 158 ± 32 s(-1)。血红素丙酸盐振动的衰减速率与再结合速率一致，这表明在远端血红素环境中的配体动力学和血红素丙酸盐感知的环境之间存在耦合。这些结果对于与近端 His-Fe 血红素环境有关的问题，包括丙酸盐和远端口袋中带正电荷或供质子的残基，对于腈的合成至关重要，讨论了这些结果的意义。
