Miller M A, Han G W, Kraut J
Department of Chemistry, University of California at San Diego, La Jolla 92093-0317.
Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11118-22. doi: 10.1073/pnas.91.23.11118.
Cytochrome c peroxidase reacts with peroxide to form compound I, which contains an oxyferryl heme and an indolyl radical at Trp-191. The indolyl free radical has a half-life of several hours at room temperature, and this remarkable stability is essential for the catalytic function of cytochrome c peroxidase. To probe the protein environment that stabilizes the compound I radical, we used site-directed mutagenesis to replace Trp-191 with Gly or Gln. Crystal structures of these mutants revealed a monovalent cation binding site in the cavity formerly occupied by the side chain of Trp-191. Comparison of this site with those found in other known cation binding enzymes shows that the Trp-191 side chain resides in a consensus K+ binding site. Electrostatic potential calculations indicate that the cation binding site is created by partial negative charges at the backbone carbonyl oxygen atoms of residues 175 and 177, the carboxyl end of a long alpha-helix (residues 165-175), the heme propionates, and the carboxylate side chain of Asp-235. These features create a negative potential that envelops the side chain of Trp-191; the calculated free energy change for cation binding in this site is -27 kcal/mol (1 cal = 4.184J). This is more than sufficient to account for the stability of the Trp-191 radical, which our estimates suggest is stabilized by 7.8 kcal/mol relative to a Trp radical in solution.
细胞色素c过氧化物酶与过氧化物反应形成化合物I，该化合物在色氨酸-191处含有一个氧合铁血红素和一个吲哚基自由基。吲哚基自由基在室温下具有数小时的半衰期，这种显著的稳定性对于细胞色素c过氧化物酶的催化功能至关重要。为了探究稳定化合物I自由基的蛋白质环境，我们使用定点诱变将色氨酸-191替换为甘氨酸或谷氨酰胺。这些突变体的晶体结构揭示了一个单价阳离子结合位点，该位点位于先前被色氨酸-191侧链占据的空腔中。将该位点与其他已知阳离子结合酶中的位点进行比较表明，色氨酸-191侧链位于一个共有钾离子结合位点中。静电势计算表明，阳离子结合位点是由175和177位残基的主链羰基氧原子、一个长α-螺旋（165-175位残基）的羧基末端、血红素丙酸酯以及天冬氨酸-235的羧酸盐侧链上的部分负电荷形成的。这些特征产生了一个包围色氨酸-191侧链的负电势；该位点中阳离子结合的计算自由能变化为-27千卡/摩尔（1卡 = 4.184焦耳）。这足以解释色氨酸-191自由基的稳定性，我们的估计表明，相对于溶液中的色氨酸自由基，它的稳定性为7.8千卡/摩尔。
