In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase-The Interplay of Key Amino Acids, PH, Buffer and Glycerol

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2021 Sep 28

PMID 34576013

Authors

Kevin Nys

Vera Pfanzagl

Jeroen Roefs

Christian Obinger

Sabine Van Doorslaer

Affiliations

Soon will be listed here.

Abstract

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from (KDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.

References

Zamocky M, Hofbauer S, Schaffner I, Gasselhuber B, Nicolussi A, Soudi M . Independent evolution of four heme peroxidase superfamilies. Arch Biochem Biophys. 2015; 574:108-19. PMC: 4420034. DOI: 10.1016/j.abb.2014.12.025. View

Yonetani T, Wilson D, Seamonds B . Studies on cytochrome c peroxidase. 8. The effect of temperature on light absorptions of the enzyme and its derivatives. J Biol Chem. 1966; 241(22):5347-52. View

Svistunenko D, Sharpe M, Nicholls P, Blenkinsop C, Davies N, Dunne J . The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study. Biochem J. 2000; 351 Pt 3:595-605. PMC: 1221398. View

Smulevich G, Mantini A, English A, Mauro J . Effects of temperature and glycerol on the resonance Raman spectra of cytochrome c peroxidase and selected mutants. Biochemistry. 1989; 28(12):5058-64. DOI: 10.1021/bi00438a024. View

Tamura M, Hori H . Optical and magnetic measurements of horseradish peroxidase. 3. Electron paramagnetic resonance studies at liquid-hydrogen and -helium temperatures. Biochim Biophys Acta. 1972; 284(1):20-9. DOI: 10.1016/0005-2744(72)90041-1. View

Smirnova T, Weber R, Davis M, Franzen S . Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite ornata: HYSCORE experiments. J Am Chem Soc. 2008; 130(7):2128-9. DOI: 10.1021/ja0772952. View

Vinck E, Van Doorslaer S, Dewilde S, Moens L . Structural change of the heme pocket due to disulfide bridge formation is significantly larger for neuroglobin than for cytoglobin. J Am Chem Soc. 2004; 126(14):4516-7. DOI: 10.1021/ja0383322. View

Aasa R, ELLFOLK N, Ronnberg M, Vanngard T . Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase. Biochim Biophys Acta. 1981; 670(2):170-5. DOI: 10.1016/0005-2795(81)90005-2. View

Garcia-Rubio I, Fittipaldi M, Trandafir F, Van Doorslaer S . A multifrequency HYSCORE study of weakly coupled nuclei in frozen solutions of high-spin aquometmyoglobin. Inorg Chem. 2009; 47(23):11294-304. DOI: 10.1021/ic8016886. View

10.

Hofbauer S, Pfanzagl V, Michlits H, Schmidt D, Obinger C, Furtmuller P . Understanding molecular enzymology of porphyrin-binding α + β barrel proteins - One fold, multiple functions. Biochim Biophys Acta Proteins Proteom. 2020; 1869(1):140536. PMC: 7611857. DOI: 10.1016/j.bbapap.2020.140536. View

11.

Linde D, Pogni R, Canellas M, Lucas F, Guallar V, Baratto M . Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study. Biochem J. 2014; 466(2):253-62. PMC: 4357238. DOI: 10.1042/BJ20141211. View

12.

Yonetani T, Anni H . Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group. J Biol Chem. 1987; 262(20):9547-54. View

13.

Hofbauer S, Schaffner I, Furtmuller P, Obinger C . Chlorite dismutases - a heme enzyme family for use in bioremediation and generation of molecular oxygen. Biotechnol J. 2014; 9(4):461-73. PMC: 4162996. DOI: 10.1002/biot.201300210. View

14.

Bizzarri A, Cannistraro S . Solvent modulation of the structural heterogeneity in FeIII myoglobin samples: a low temperature EPR investigation. Eur Biophys J. 1993; 22(4):259-67. DOI: 10.1007/BF00180260. View

15.

Svistunenko D, Worrall J, Chugh S, Haigh S, Ghiladi R, Nicholls P . Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH. Biochimie. 2012; 94(6):1274-80. DOI: 10.1016/j.biochi.2012.02.021. View

16.

Howes B, Rodriguez-Lopez J, Smith A, Smulevich G . Mutation of distal residues of horseradish peroxidase: influence on substrate binding and cavity properties. Biochemistry. 1997; 36(6):1532-43. DOI: 10.1021/bi962502o. View

17.

Goblirsch B, Kurker R, Streit B, Wilmot C, DuBois J . Chlorite dismutases, DyPs, and EfeB: 3 microbial heme enzyme families comprise the CDE structural superfamily. J Mol Biol. 2011; 408(3):379-98. PMC: 3075325. DOI: 10.1016/j.jmb.2011.02.047. View

18.

Peisach J, Blumberg W, Ogawa S, Rachmilewitz E, Oltzik R . The effects of protein conformation on the heme symmetry in high spin ferric heme proteins as studied by electron paramagnetic resonance. J Biol Chem. 1971; 246(10):3342-55. View

19.

Smulevich G, Feis A, Howes B . Fifteen years of Raman spectroscopy of engineered heme containing peroxidases: what have we learned?. Acc Chem Res. 2005; 38(5):433-40. DOI: 10.1021/ar020112q. View

20.

Blumberg W, Peisach J, Wittenberg B, Wittenberg J . The electronic structure of protoheme proteins. I. An electron paramagnetic resonance and optical study of horseradish peroxidase and its derivatives. J Biol Chem. 1968; 243(8):1854-62. View