Peroxidase Catalytic Cycle of MCM-41-Entrapped Microperoxidase-11 as a Mechanism for Phenol Oxidation

Abstract

The encapsulation of microperoxidases (MPs) into molecular sieves with controlled pore size, such as the mesoporous silica MCM-41, represents a nanotechnology strategy to control the catalytic properties of MPs and mimic the enzymatic activity of hemoproteins. In this work, the ferric microperoxidase-11 (MP-11), obtained from trypsin-catalyzed hydrolysis of horse-heart cytochrome c, was entrapped in MCM-41, thus resulting in a catalyst (Fe(III)MP11MCM41) with catalase and monooxygenase properties. The entrapment of MP-11 inside MCM-41 was confirmed by elemental analysis and UV-visible spectrum, with a red shift in the Soret band indicating that the heme group was in a hydrophobic microenvironment. Similarly to catalase, the catalyst Fe(III)MP11MCM41 exhibited specificity for hydrogen peroxide to be converted to a high-valence oxidized intermediate, Compound II. Also mimicking catalase, the cleavage of hydrogen peroxide by MP11MCM41 resulted in O2 production detected by a Clark electrode. Phenol was able to act as reducing agent of MP11MCM41 Compound II leading to the completion of a peroxidase cycle, as confirmed by UV-visible spectrometry and EPR measurements. The analysis of the reaction products by high performance liquid chromatogram coupled to tandem mass spectrometry (HPLC/MS) revealed 2,4-dihydroxyphenol as the product of phenol oxidation by MP11MCM41. Therefore, in addition to catalase activity, the catalyst MP11MCM41 also displayed monooxygenase properties, which was possible because the MP-11 heme iron promotes homolytic cleavage of the hydrogen peroxide generating hydroxyl radicals. With such characteristics, MCM-41-entrapped MP-11 is a promising catalyst for nanobiotechnological devices.

Enhancing microperoxidase activity and selectivity: immobilization in metal-organic frameworks

“…[43] Such materials have been applied to many enzymes, but very rarely to MPs. [44][45][46] MPs have mainly been adsorbed or covalently bound onto surfaces that provided biodevices with interesting properties, but they did not provide protective environments to MPs. [11,[47][48][49] This may be due to the nature of the materials.…”

Technological Applications of Porphyrins and Related Compounds: Spintronics and Micro-/Nanomotors

“…The remarkable chemical and photophysical properties of porphyrins have attracted the interest of researchers worldwide [1]. Biological and technological applications of porphyrins can involve the use of native hemeproteins, metallo-substituted hemeproteins, and the product of the tryptic digestion of horse heart cytochrome c, microperoxidases [11][12][13][14][15][16][17][18][19]. Inspired by nature, researchers have synthesized a diversity of nonnatural porphyrins.…”

Nanostructures for peroxidases

“…The water-soluble heme-containing peptides obtained by proteolytic digestion of cytochrome c are the microperoxidases (MP) often used to explore aspects of the chemistry of iron porphyrins, and as mimics for some reactions catalyzed by peroxidases (Prieto et al, 2006 ; Araujo et al, 2007 ; Marques, 2007 ). MP are not only model compounds but also useful molecules for applications in biosensors as electron carriers, photoreceptors, microzymes, and drugs.…”

“…MP-11, a MP with 11 aminoacids residues, assembles to boron nitride nanotubes which enhance catalysis due to a strong electron coupling between the active center of MP-11 and the nanotube showing that nanostructures often modulate peroxidases activity (Li et al, 2014b ). The assembly of peroxidases into micelles (Prieto et al, 2006 ), reversed micelles (Das and Das, 2009 ; Maiti et al, 2012 ; Das et al, 2013 ), liposomes (Dotsikas and Loukas, 2012 ), supported bilayers (Leão-Silva et al, 2011 ), lipid monolayers at the air-water interface (Schmidt et al, 2008 ), organic, inorganic, metallic, magnetic or composite nanoparticles (Xu and Han, 2004 ; Kim et al, 2005 ; Araujo et al, 2007 ; Khaja et al, 2007 ; Silva et al, 2007 ; Chen et al, 2011 ; Klyachko et al, 2012 ; Li et al, 2012 ; Pan et al, 2013 ; Duan et al, 2014 ), hydrogels and microgels (Nakashima et al, 2009 ; González-Sánchez et al, 2011 ; Wu et al, 2012 ; Bruns et al, 2013 ), nanotubes (Li et al, 2014b ), multilayers and nanostructured films (Kim et al, 2010 ; Pallarola et al, 2012 ; Cortez et al, 2013 ) has been explored for important applications such as immunoassays design (Marquette and Blum, 2009 ; Dotsikas and Loukas, 2012 ), drug delivery (Allen et al, 2011 ; Kotchey et al, 2013 ), cancer therapy (Ibañez et al, 2015 ), environment protection and bioremediation (Bansal and Kanwar, 2013 ; Duan et al, 2014 ), construction of sensors (Xu and Han, 2004 ; Kim et al, 2005 ; Chen et al, 2011 ; Pallarola et al, 2012 ; Chen and Chatterjee, 2013 ; Cortez et al, 2013 ), and energy production (Ramanavicius et al, 2005 ; Ramanavicius and Ramanaviciene, 2009 ). HRP adsorbs onto silicon wafers providing reusable films for emulsion polymerization (Naves et al, 2007 ) and is useful for tissue engineering applications by forming hydrogels in situ via crosslinking (Bae et al, 2014 ) and for atom transfer radical polymerization (Bruns et al, 2013 ).…”