Isogai Y, Iizuka T, Shiro Y
Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
J Biol Chem. 1995 Apr 7;270(14):7853-7. doi: 10.1074/jbc.270.14.7853.
Phagocytic cytochrome b558 is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction kinetics of cytochrome b558 purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b558 was rapidly reoxidized by O2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b558 with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O2 linearly depended on the O2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pKa of 9.7. Under complete anaerobic conditions, ferrous cytochrome b558 was oxidized by ferricyanide at a rate faster than by O2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b558 are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O2 complex. These findings are most consistent with the electron transfer from the heme to O2 by an outer-sphere mechanism.
吞噬细胞色素b558是一种独特的含血红素酶，它催化分子氧的单电子还原，以产生具有六配位血红素铁的超氧阴离子。为了阐明超氧产生的机制，我们通过停流和快速扫描光谱分析了从猪中性粒细胞中纯化的细胞色素b558的氧化还原动力学。还原型细胞色素b558被O2迅速再氧化，显示出具有清晰等吸收点的光谱变化，在厌氧条件下用连二亚硫酸钠还原铁细胞色素b558的过程中也观察到了这种变化。与O2反应的单周转速率线性依赖于O2浓度，但不受CO添加的影响。反应速率随着pH值的升高而降低，pKa为9.7。在完全厌氧条件下，亚铁细胞色素b558被铁氰化物氧化的速率比被O2氧化的速率快。热力学分析表明，与通过形成铁-O2复合物的天然和修饰肌红蛋白的自氧化相比，细胞色素b558反应的焓能垒显著更低。这些发现与通过外层球机制从血红素到O2的电子转移最为一致。
