A DyP-type peroxidase at a bio-compatible interface: structural and mechanistic insights

Abstract

Direct electronic coupling of peroxidases with bio-compatible interfaces allows for investigation of enzyme's electro-catalytic properties that are essential in the design of bio-electronic devices. Here, a novel dye decolourising-type peroxidase from Pseudomonas putida MET94 (PpDyP) is immobilised on Ag electrodes coated with an alkanethiol self-assembled monolayer. Structural features of the active site, heterogeneous electron transfer and electro-catalytic properties of immobilised PpDyP are addressed by combination of surface enhanced spectroscopic and electrochemical approaches. They reveal that the structural integrity of the heme pocket of PpDyP is preserved upon immobilisation, the enzyme is electronically coupled to the electrode, and it exhibits efficient catalytic activity. Importantly, no significant modulation of the midpoint redox potential (E m ) of the immobilised protein (E m À300 mV) is observed with respect to that in solution (E m,sol À260 mV). This study provides important structural and mechanistic insights into immobilised DyP-type peroxidase, capable of efficient decolourisation of numerous dyes, revealing PpDyP as a promising candidate for biotechnological applications.

Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

“…This assignment is consistent with the frequencies reported in the literature for catalytic intermediates of classical peroxidases. [11][12][13][14][15][16] In the absence of the reference RR spectra of Cpd I and Cpd II in DyPs, the assignment of the 1378 cm À1 and 1508 cm À1 modes to the oxyferryl Fe(IV)QO porphyrin p cation is supported by the evidence from our previous work: (i) electronic absorption spectra, which reveal a fingerprint of stable Cpd I in the reaction of PpDyP with 1-3 equivalents of H 2 O 2 in solution; [8][9][10] 8 The n 4 /n 3 values that we observe here are slightly higher than those reported for Cpd I in the literature, 11 which may be an intrinsic property of DyPs, or an indication of Cpd II. For instance, n 4 and n 3 modes of Cpd I in HRP were found at 1373-1376 and 1505 cm À1 , respectively, but at 1379 and 1510 cm À1 in Cpd II.…”

“…9,10 Component analysis of the spectra reveals well resolved bands of the n 4 , n 3 , n 2 , n 10 and n CQC modes at 1376, 1502, 1572, 1626 and 1637 cm À1 (Fig. 1a, green trace) and at 1373, 1494, 1564, 1621 and 1632 cm À1 (Fig.…”

“…solution vs. crystalline form) induce a typically reversible inter-conversion of the spin populations. 9,10,18,19 Upon addition of 1 equivalent of hydrogen peroxide to the SERR cell holding the enzyme-loaded metal support, well reproducible spectral changes occur instantaneously: a pronounced decrease of signal intensity, accompanied by an upshift of n 4 and n 3 modes and a broadening of n 2 , n 10 and n CQC bands (Fig. S1, ESI †).…”

“…[1][2][3][4][5][6][7][8][9][10] They lack the distal histidine that is highly conserved in classical peroxidases and which acts as an acid/base catalyst in the reduction of hydrogen peroxide to water. DyPs are classified into four phylogenetically distinct classes (A-D); their physiological role is not fully established yet and it appears to be subfamilydependent.…”

“…1a, blue trace), which are attributed to quantum mechanically mixed-spin (QS) and 5-coordinated high spin (5cHS) species, respectively. 9,10 In addition, small contributions of a 6-coordinated HS (6cHS) species with n 4 at 1366 cm À1 and n 3 at 1483 cm À1 are also present in the spectra. The SERR spectra of PpDyP immobilised on Ag supports coated with mixed amino-and hydroxyl-terminated alkanethiol self-assembled monolayers (SAM), employing aminooctanethiol (AOT) and mercaptohexanol (MOH) in a 1 : 3 (M/M) ratio, reveal essentially the same band frequencies as RR spectra ( Fig.…”