Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

Abstract

Using oligonucleotide-directed site-specific mutagenesis, we have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 degrees C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

“…Protein Production and Purification-Purification of CcP was undertaken using a modified literature protocol (55,56). 4 ϫ 20 ml of LB containing 100 g/ml ampicillin were inoculated with one colony from an L-agar plate containing 100 g/ml ampicillin streaked from a glycerol stock of Escherichia coli BL21-CODONPLUS(DE3)-RIL[pT7CCP-Zf1] (56).…”

Higher oxidation states of prostaglandin H synthase

“…In this reaction PGG2 was not a reactant as was shown by comparing the dependence of the reaction rate on PGG2 concentration with the kinetic models. The electronic spectrum of this intermediate resembled compound I1 of horseradish peroxidase [I41 or complex ES of cytochrome-c peroxidase [18], structures which have been assigned to [(protoporphyrin IX)Fe"OH]. The question of whether intermediate I1 was a one-electron or two-electron oxidized state compared to the native enzyme can be answered from the findings that (a) PGGz was not a reactant for the reaction of intermediate I to intermediate 11, (b) there were no exogenous electron donors present, and (c) a tyrosyl radical has been detected by EPR concomitantly with the electronic spectrum of intermediate I1 [19].…”

“…In contrast, the Fe(V) state of cytochrome-c peroxidase, complex ES, has the structure [(protoporphyrin IX)FelvO]R' [17], where the radical R' from an amino acid side-chain has not been identified yet [18]. In the Fe(1V) state of horseradish peroxidase, compound 11, the porphyrin n-cation radical has received an electron yielding the structure [(protoporphyrin 1X)Fe"OHj.…”

Protein engineering. The design, synthesis and characterization of factitious proteins

“…The longer range class of electron transfers typified by the interactions of cytochrome c with either cytochrome c peroxidase or cytochrome b5 are cases in point. The former redox pair has been under intensive study by several groups and use has been made of both mutational changes (Goodin et al, 1986;Fishel et al, 1987), and the replacement of iron by zinc in one of the pair (McClendon et al, 1985;Liang et al, 1987). Under conditions wherein the donor-acceptor complex is preformed and stable it is possible to excite the zinc protoporphyrin and observe a unidirectional first-order electron transfer step to the ferri-haem partner.…”

“…Site-directed mutagenesis of cytochrome c peroxidase (Fishel et al, 1987) has been used to test the hypothesis that Trp-51 constitutes the locus of the first free radical observed in the reduction of hydroperoxides for cytochrome c in the presence of its peroxidase. The position of the indole Trp-51, some 0.33 nm above and nearly co-planar with the haem, seemed to be a compelling argument for its involvement.…”