Watmough N J, Cheesman M R, Greenwood C, Thomson A J
Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich, U.K.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):469-75. doi: 10.1042/bj3000469.
Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.
氧化型细胞色素 bo 能与微摩尔浓度的过氧化氢迅速反应，形成单一衍生物。该化合物的电子吸收光谱与本实验室报道的该酶氧化形式的光谱不同[瓦特莫、切斯曼、根尼斯、格林伍德和汤姆森（1993 年），《欧洲生物化学学会联合会快报》319 卷，151 - 154 页]。其特征在于在 411 nm 处有一个 Soret 最大值，在 555 nm 处吸光度增加，在 624 nm 处强度降低。此过程的表观解离常数约为 4×10⁻⁶ M，形成新化合物的双分子速率常数为(1.25 - 1.7)×10³ M⁻¹·s⁻¹。电子吸收差光谱表明该产物与酶的混合价形式与双氧反应形成的化合物相同。通过室温磁圆二色光谱对该化合物进行研究表明，血红素 o 既不是高自旋也不是低自旋铁离子，而是具有氧合铁（IV）物种的特征光谱。没有证据表明卟啉环被氧化。因此，该物种的双核中心必定由一个氧合铁（IV）血红素（S = 1）与一个 Cu(II)离子（S = 1/2）耦合形成一个新的顺磁中心。该反应也通过 X 波段电子顺磁共振光谱进行跟踪，结果表明与氧合铁（IV）物种的形成同时消失的是氧化酶中弱耦合双核中心产生的宽 g = 3.7 的信号。由于未观察到新的可通过电子顺磁共振检测到的顺磁物种，推测 Cu(II)离子与另一个顺磁体耦合，可能是一个有机自由基。电子吸收光谱中没有证据表明细胞色素 bo 与过氧化氢进一步反应形成第二种物质。我们认为这种氧合铁（IV）物种的形成情况与细胞色素 c 氧化酶的 P 形式相同，P形式通常被认为含有一个结合的过氧离子。讨论了这些观察结果对血红素 - 铜末端氧化酶反应机制的影响。
