Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
J Am Chem Soc. 2010 Apr 28;132(16):5711-24. doi: 10.1021/ja9082182.
Chlorite dismutase catalyzes O(2) release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady-state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3-11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid-base transition was observed by UV/visible and resonance Raman (rR) spectroscopy with a pK(a) of 8.7, 2-3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five-coordinate high-spin Fe(III) to a mixed high/low-spin ferric hydroxide, as confirmed by rR spectroscopy. The two Fe-OH stretching frequencies are quite low, consistent with a weak Fe-OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe-OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond-donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pK(a) approximately 6.5) form also appears to act as the active-site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady-state profile for the rate of chlorite decomposition is characterized by these same pK(a) values. The five-coordinate high-spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum k(cat) = 2.0(+/-0.6) x 10(5) s(-1), k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1), pH 5.2, 4 degrees C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.
亚氯酸盐歧化酶能高效且特异地催化亚氯酸盐释放氧气。为了深入了解环境中质子化状态对形成二氧化氧的影响，本研究对来自 Dechloromonas aromatica 的亚氯酸盐歧化酶的光谱性质、配体结合亲和力和稳态动力学进行了 pH 3-11.5 范围内的检测。通过紫外/可见和共振拉曼（rR）光谱观察到酸碱转换，其 pK(a) 值为 8.7，比典型的 His 配位过氧化物酶中观察到的类似转换低 2-3 pH 单位。此转换标志着五配位高自旋 Fe(III)转化为混合高/低自旋的铁氢氧化物，这通过 rR 光谱得到证实。两个 Fe-OH 伸缩频率非常低，与近中性咪唑侧链的近端组氨酸配体一致，表明 Fe-OH 键较弱。尽管如此，由于远端的 H-bond 供体的相互作用，羟基仍强烈存在，从而削弱了 Fe-OH 键。随着 pH 的变化，Cld-CO 的 rR 光谱揭示了该复合物的两种形式，一种形式中，远端残基与羰基氧的相互作用最小，另一种形式则为酸性形式，其中氧受到正电荷的影响。最近的晶体学数据表明，183 位精氨酸是远端口袋中唯一的 H-bond 供体残基。很可能是这种带正电荷的 Arg 充当了振动数据所暗示的与外源轴向血红素配体相互作用的强正 H-bond 供体。在结合质子化配体（如 HCN）与 ferric Cld 的反应中，同样处于中性（pK(a)约 6.5）形式的 Arg 似乎也充当了活性位点碱。该稳态氯离子分解速率的特征由相同的 pK(a) 值确定。五配位高自旋的酸性 Cld 比碱性羟基结合形式更活跃。当远端的 Arg 质子化/带正电荷（最大 k(cat) = 2.0(+/-0.6) x 10(5) s(-1)，k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1)，pH 5.2，4 摄氏度）时，酸性形式分解亚氯酸盐的效率最高，当它在碱性条件下充当轴向氢氧化物配体的 H-bond 供体时，其效率略有降低。
