Engineering ascorbate peroxidase activity into cytochrome c peroxidase

Abstract

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.

Fig. 1

WTCCP (red) superimposed on APX (green). Panels A and B are two different views.

Fig. 2

The engineered ascorbate binding loop in CCP2APX. (A) 2F_o−F_c electron density map contoured at 1.0 σ. Note that the engineered loop centered on Lys33 and Arg 172 which is Asn in WTCCP are very well ordered. (B) A superimposition of CCP2APX/F191 (red) on the APX-ascorbate complex (green). The key components of the ascorbate binding site are the same in both enzymes.

Fig. 3

Average MD structures (magenta) superimposed on original structures (green) for (A) APX, (B) CCP2APX and (C) CCP2APX with Asn 87 converted to Ile. Note that in APX (panel A) the ascorbate remains stable throughout the MD simulations while in CCP2APX (panel B) the ascorbate moves up toward Asn 87 thus allowing the ascorbate and Asn80 to form an H-bond. The movement of the substrate results in the loss of the heme-ascorbate H-bond. The in silico conversion of Asn 87 to Ile in CCP2APX results in the ascorbate maintaining its interactions with Arg 184 and the heme.

Fig. 4

1.06 Å 2F_o−F_c maps for the CCP/R184 mutant contoured at 2.5 σ in panels A, B, and C and 5.0 σ in panel D. A) The region around the mutant side chain Arg 184. B) The distal pocket showing the conserved residues Arg 48 and His 52 that are the catalytic groups responsible for heterolytic cleavage of the peroxide O–O bond and formation of compound I. C) The proximal binding pocket showing the conserved His175 ligand and its H-bonding partner Asp235. Trp 191 is the site of free radical formation in compound I. D). The 2F_o−F_c map contoured at 5.0 σ after 10 rounds of refinement with no angle or distance restraints applied to Asp 235. Note the continuous density along the CG-OD2 bond suggesting a double bond while the weaker connectivity between CG and OD1 indicates a single bond.

Fig. 5

A) UV-vis spectra of CCP2APX Fe³⁺ resting state (blue), immediately after the addition of 1 equivalent of H₂O₂ to give compound I (red), and one hour after the addition of H₂O₂ (green). B) EPR spectrum of CCP2APX immediately after the addition of 1 equivalent of H₂O₂.

Fig. 6

A) Diode-array spectrum of CCP2APX/F191 before (labeled as Fe³⁺ and 6 ms after mixing (labeled as compound I) with H₂O₂. B) UV/Vis spectrum of CCP2APX/F191 before (blue), immediately after the addition of 1 equivalent of H₂O₂ (red), and about 30 seconds after the addition of H₂O₂ (green).

Fig. 7

Ascorbate peroxidase activity of CCP2APX. 1.0 µM enzyme was added to a cuvette containing 250 µM H₂O₂ and varying concentrations of ascorbate. The rate was determined by the decrease in absorbance at 290 nm using a ε₂₉₀ = 2.8 mM⁻¹ cm⁻¹.