Silvestrini M C, Colosimo A, Brunori M, Walsh T A, Barber D, Greenwood C
Biochem J. 1979 Dec 1;183(3):701-9. doi: 10.1042/bj1830701.
Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.
对铜绿假单胞菌细胞色素氧化酶样品进行铁含量测定和干重测量，并结合对天然蛋白质和共价交联寡聚体的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳研究，以估算该酶的分子量和光谱吸收系数。对于总分子量为122000（基于铁分析）的二聚体蛋白质分子，计算出ε(ox.)(410)=282×10³升·摩尔⁻¹·厘米⁻¹。对亚硝酸催化还原型天青蛋白氧化反应的稳态动力学观察表明，随着pH从5.7升高到7.2，酶失活显著增加。由于已知铜绿假单胞菌细胞色素氧化酶的亚硝酸还原酶活性产物NO会与该酶结合，因此开展了一项研究以评估NO作为产物抑制剂的可能性。研究表明，pH值为4、5和6时的氧化型蛋白质样品，NO会与血红素c和d(1)成分结合，但pH值为7及以上的氧化型酶样品在置于该气体氛围下时会形成其还原型配体结合形式。还发现pH值为4、5、6和7时抗坏血酸还原的酶样品在两个血红素成分上均能结合NO，不过在pH值为7时血红素c的结合速率非常缓慢。在pH值为8和9时，只有亚铁血红素d(1)能结合NO。在pH值5 - 7范围内用亚硝酸作为NO的前体对还原型蛋白质进行滴定实验表明，血红素d(1)的亲和力比血红素c高得多：在pH值5.2和5.9时用NO平衡溶液对氧化型酶进行的实验也显示出相同的行为模式。
