Newmyer S L, Sun J, Loehr T M, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1996 Oct 1;35(39):12788-95. doi: 10.1021/bi9609331.
The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.
辣根过氧化物酶（HRP）中近端铁配体是His-170。多聚组氨酸标记的HRP（hHRP）的H170A突变体已在杆状病毒系统中表达，并已进行纯化和表征。在pH 7时，该突变体的Soret峰在414 nm处，而不是403 nm。共振拉曼光谱表明，该蛋白质在铁离子状态下主要是六配位低自旋，亚铁状态下在212 cm-1处有一个谱带，表明远端组氨酸与铁配位。外源咪唑（Im）以Kd = 22 +/- 4 mM的亲和力与该酶结合。H170A hHRP与H2O2反应不会产生光谱可检测的化合物I或化合物II中间体，但会导致血红素基团逐渐降解。然而，H170A hHRP具有催化活性，在饱和浓度的Im存在下，其愈创木酚和ABTS过氧化物酶活性分别提高了260倍和125倍。Im对愈创木酚和ABTS的刺激作用的Km均为24 mM。H170A hHRP的pH谱与野生型hHRP不同，但这些差异在Im存在下基本消除。通过稳态动力学方法测定，H170A hHRP形成“化合物I”的速率在无Im时为k1 = 16 M-1 s-1，在有Im时为k1 = 2.4 x 10(4) M-1 s-1。野生型hHRP的相应速率为k1 = 4.4 x 10(6) M-1 s-1。结果表明，Im结合在由H170A突变产生的空腔中，与血红素铁原子配位，并通过挽救化合物I的形成速率恢复了大部分催化活性。然而，Im对催化活性的这种挽救可能受到H170A突变体中血红素与远端组氨酸（His-42）配位的限制。因此，近端组氨酸的主要功能是将铁原子固定，以不利于第六配体结合，特别是铁与远端组氨酸的配位。此外，近端配体的强氢键对于促进化合物I形成过程中的O - O键断裂可能至关重要。
