Santoni Elisa, Jakopitsch Christa, Obinger Christian, Smulevich Giulietta
Dipartimento di Chimica, Universitá di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy.
Biochemistry. 2004 May 18;43(19):5792-802. doi: 10.1021/bi035835b.
A detailed resonance Raman and electronic absorption investigation has been carried out on a series of novel distal and proximal variants of recombinant catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. In particular, variants of the distal triad Pro-Asp-Asn and the proximal triad His-Asp-Trp have been studied in their ferric and ferrous states at various pH. The data suggest marked differences in the structural role of the conserved residues and hydrogen-bond networks in KatG and CCP, which might be connected to the different catalytic activity. In particular, in KatG the proximal residues have a major role in the stability of the protein architecture because the disruption of the proximal Trp-Asp hydrogen bond by mutation weakens heme binding to the protein. On the distal side, replacing the hydrogen-acceptor carboxamide group of Asn153 by an aspartate carboxylate group or an aliphatic residue alters or disrupts the hydrogen bond with the distal His. As a consequence, the basicity of His123 is altered. The effect of mutation on Asp152 is noteworthy. Replacement of the Asp152 with Ser makes the architecture of the protein very similar to that of CCP. The Asp152 residue, which has been shown to be important in the hydrogen peroxide oxidation reaction, is expected to be hydrogen bonded to the nitrogen atom of Ile248 which is part of the KatG-specific insertion LL1, as in other KatGs. This insertion is at one edge of the heme, and connects the distal side with the proximal helices E and F, the latter carrying the proximal His ligand. We found that the distal Asp-Ile hydrogen bond is important for the stability of the heme architecture and its alteration changes markedly the proximal His-Asp hydrogen-bond interaction.
对来自集胞藻PCC 6803的重组过氧化氢酶-过氧化物酶的一系列新型远端和近端变体进行了详细的共振拉曼和电子吸收研究。特别是，对远端三联体Pro-Asp-Asn和近端三联体His-Asp-Trp的变体在不同pH值下的铁态和亚铁态进行了研究。数据表明，KatG和CCP中保守残基和氢键网络的结构作用存在显著差异，这可能与不同的催化活性有关。特别是，在KatG中，近端残基在蛋白质结构的稳定性中起主要作用，因为通过突变破坏近端Trp-Asp氢键会削弱血红素与蛋白质的结合。在远端，用天冬氨酸羧酸盐基团或脂肪族残基取代Asn153的氢受体羧酰胺基团会改变或破坏与远端His的氢键。结果，His123的碱性发生改变。Asp152突变的影响值得注意。用Ser取代Asp152使蛋白质结构与CCP非常相似。如在其他KatG中一样，已证明在过氧化氢氧化反应中起重要作用的Asp152残基预计与Ile248的氮原子形成氢键，Ile248是KatG特异性插入片段LL1的一部分。该插入片段位于血红素的一侧边缘，将远端与近端螺旋E和F连接起来，后者携带近端His配体。我们发现远端Asp-Ile氢键对血红素结构的稳定性很重要，其改变会显著改变近端His-Asp氢键相互作用。
