Cutruzzolà Francesca, Arese Marzia, Ranghino Graziella, van Pouderoyen Gertie, Canters Gerard, Brunori Maurizio
Dipartimento di Scienze Biochimiche, Università di Roma La Sapienza, P. le A. Moro 5, 00185 Rome, Italy.
J Inorg Biochem. 2002 Feb;88(3-4):353-61. doi: 10.1016/s0162-0134(01)00390-7.
Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.
来自铜绿假单胞菌的细胞色素c(551)是一种由82个氨基酸残基组成的单体氧化还原蛋白，作为cd(1)亚硝酸还原酶的生理电子供体参与异化反硝化作用。c(551)表面带电残基的分布非常不均匀：一侧富含酸性残基，而另一侧则有一圈主要为赖氨酸的正侧链，位于围绕血红素裂隙的疏水斑块边界。为了确定细胞色素c(551)中参与电子转移的表面区域，我们对属于疏水斑块的三个残基进行了特异性突变，即Val23→Asp、Pro58→Ala和Ile59→Glu。通过研究自交换速率以及对铜绿假单胞菌cd(1)亚硝酸还原酶（其生理伴侣）和铜绿假单胞菌天青蛋白（一种常用于体外模型氧化还原伴侣的铜蛋白）的电子转移活性，分析了这些突变的影响。我们的结果表明，在疏水斑块中引入负电荷会严重阻碍同核和异核电子转移。
