Kinetics of Î±-MnOOH Nanoparticle Formation through Enzymatically

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Kinetics of #-MnOOH Nanoparticle Formation through Enzymatically-catalyzed Biomineralization inside Apoferritin Yue Hui, Haesung Jung, Doyoon Kim, and Young-Shin Jun Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00568 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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Crystal Growth & Design

Kinetics of -MnOOH Nanoparticle Formation through Enzymatically-catalyzed Biomineralization inside Apoferritin Yue Hui,† Haesung Jung,† Doyoon Kim, and Young-Shin Jun* Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, Missouri 63130, United States †

These authors contributed equally *To whom correspondence should be addressed

Abstract While biomineralization in apoferritin has effectively synthesized highly monodispersed nanoparticles of various metal oxides and hydroxides, the detailed kinetics and mechanisms of Mn(III) (hydr)oxide formation inside apoferritin cavities have not been reported. To address this knowledge gap, we first identified the phase of solid Mn(III) formed inside apoferritin cavities as -MnOOH. To analyze the oxidation and nucleation mechanism of -MnOOH inside apoferritin by quantifying oxidized Mn, we used a colorimetric method with leucoberbelin blue (LBB) solution. In this method, LBB dissembled apoferritin by inducing an acidic pH environment, and reduced MnOOH nanoparticles. The LBB-enabled kinetic analyses of -MnOOH nanoparticle formation suggested that the orders of reaction with respect to Mn2+ and OH- are 2 and 4, respectively, and -MnOOH formation follows two-step pathways: First, soluble Mn2+ undergoes apoferritin catalyzed oxidation at the ferroxidase dinuclear center, forming a Mn(III)-protein complex, P[Mn2O2(OH)2]. Second, the oxidized Mn(III) dissociates from the protein binding sites and are subsequently nucleated to form -MnOOH nanoparticles in the apoferritin cavities. This study reveals key kinetics and mechanistic information of the Mn-apoferritin systems and the results facilitate applications of apoferritin as a means of nanomaterial synthesis.

Young-Shin Jun, Ph.D., Professor Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130 Phone: (314)-935-4539 Fax: (314)-935-7211 E-mail: [email protected]

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Kinetics of -MnOOH Nanoparticle Formation through Enzymatically-catalyzed Biomineralization inside Apoferritin

Yue Hui,† Haesung Jung,† Doyoon Kim, and Young-Shin Jun*

Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130

E-mail: [email protected] http://encl.engineering.wustl.edu/ Submitted: August 2017


†

These authors contributed equally

*To whom correspondence should be addressed

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ABSTRACT

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While biomineralization in apoferritin has effectively synthesized highly monodispersed

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nanoparticles of various metal oxides and hydroxides, the detailed kinetics and

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mechanisms of Mn(III) (hydr)oxide formation inside apoferritin cavities have not been

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reported. To address this knowledge gap, we first identified the phase of solid Mn(III)

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formed inside apoferritin cavities as -MnOOH. To analyze the oxidation and nucleation

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mechanism of -MnOOH inside apoferritin by quantifying oxidized Mn, we used a

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colorimetric method with leucoberbelin blue (LBB) solution. In this method, LBB

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dissembled apoferritin by inducing an acidic pH environment, and reduced -MnOOH

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nanoparticles. The LBB-enabled kinetic analyses of -MnOOH nanoparticle formation

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suggested that the orders of reaction with respect to Mn2+ and OH- are 2 and 4, respectively,

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and -MnOOH formation follows two-step pathways: First, soluble Mn2+ undergoes

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apoferritin catalyzed oxidation at the ferroxidase dinuclear center, Mn(III)-protein

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complex, P-[Mn2O2(OH)2]. Second, the oxidized Mn(III) dissociates from the protein

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binding sites and are subsequently nucleated to form -MnOOH nanoparticles in the

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apoferritin cavities. This study reveals key kinetics and mechanistic information of the Mn-

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apoferritin systems and the results facilitate applications of apoferritin as a means of

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nanomaterial synthesis.



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Introduction

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Self-assembled protein cages and viral capsids have gained wide interest from

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materials scientists. Because of their shell-like structures with hollow interior space for

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bio-mineralization, they can serve as nanoreactors in the synthesis of a variety of highly

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monodispersed, inorganic nanomaterials.1-8 The variety of protein cage architectures

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allows precise control of the sizes and shapes of particles without using toxic organic

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surfactants, and results in nanoparticles with superior biocompatibility and new

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functionalities.9-12 Given these apparent advantages, extensive efforts have been made to

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establish a library of nanoreactors for synthesis of nanomaterials with specific properties.1,

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3-6, 13-17

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Cornelissen et al. generated monodisperse Prussian blue particles with a diameter of 18 ±

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1.7 nm through photocatalysis.18 These synthesized protein-virus bio-hybrids were found

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to self-organize in monolayer fashion on mica and other hydrophilic graphite surfaces.18

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In addition, Watanabe et al. reported the synthesis of Pd nanoclusters in ferritin cavities

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through reduction of Pd2+ by NaBH4.19 Compared to Pd particles alone, the Pd-

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encapsulated ferritin demonstrated a superior ability to catalyze size-selective

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hydrogenation of olefins.

For example, utilizing cowpea chlorotic mottle virus (CCMV) as a cage template,

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Ferritin, one of the most studied protein cages for nanoparticle synthesis, comprises

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a class of iron storage proteins ubiquitously present among living species.20, 21 Ferritin

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serves as an iron reservoir by sequestering iron when the cellular iron supply is high.20-22

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With its architecture of a hollow spherical shell with an outer diameter of 12 nm and a

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central cavity 8 nm in diameter, a ferritin molecule can store up to 4500 iron atoms in the

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form of Fe(III) (oxy)hydroxide through sequential oxidation and mineralization

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processes.20, 21 Ferritin is composed of 24 subunits of two types, heavy (H) and light (L).23-

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25

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in catalyzing the oxidation of Fe2+. Although lacking the catalytic site for oxidation, the L

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subunit is more efficient in the subsequent mineralization of Fe(III) because of the strong

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negative charges carried by the glutamate clusters that serve as the nucleation sites for

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mineral formation.23-25 Kinetic studies of Fe(III) (hydr)oxide formation inside apoferritin

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have revealed two iron concentration-dependent mechanisms for the overall iron oxidation

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and nucleation processes.20, 26-29 Under low iron influx (less than 48 Fe(II) atoms/protein),

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oxidation of Fe2+ to Fe3+ occurs predominantly at the ferroxidase center, where the

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enzymatic sites bind two Fe(II), and hence catalyze their oxidation in the reaction with

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dissolved oxygen, producing H2O2. As iron flux increases (more than 48 Fe(II)

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atoms/protein) and ferroxidase sites become saturated with Fe(II), Fe(II) oxidation and

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sequential Fe(III) hydrolysis occur directly on the pre-nucleated Fe(III) (oxy)hydroxide

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surface.20, 26-29

The H subunit, with a conserved dinuclear ferroxidase center, exhibits enzymatic activity

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Since the mechanism of iron deposition in native ferritin has been revealed, many

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studies have attempted to use the empty ferritin shell (apoferritin) as a macromolecular

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template for synthesis of a variety of inorganic nanoparticles, including oxides or

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hydroxides of cobalt,30 chromium,16 nickel,16 and indium,31 cadmium selenide,32 and

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cobalt/platinum alloys.33 The synthesized particles, together with the protein cage, have

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been employed in a variety of biomedical applications, such as drug delivery.34-39 The

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synthesis of Mn-apoferritin nanocomposite, in particular, has attracted great interest from

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the materials science field because of its use as a highly-functioning contrast agent in

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MRI.40-42 Mann’s group was the first to observe the successful synthesis of MnOOH

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nanoparticles in a ferritin cavity.43, 44 To elucidate the effect of apoferritin composition on

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MnOOH synthetic process, in their studies, they investigated recombinant apoferritin

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molecules with varied proportions of H- and L-chain subunits. In making their successful

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observations of MnOOH formation in apoferritin, Mann’s group correlated spectroscopic

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measurement at 450 nm with Mn(III) (oxy) hydroxide core formation inside apoferritin. In

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their study, the concentrations of solid Mn(III) formed inside apoferritin cavities could not

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be specified, and thus the detailed kinetics and mechanism of Mn(II) oxidation and Mn(III)

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(oxy) hydroxide nucleation in apoferritin were not reported. To better control of material’s

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properties, however, it is important to have sufficient knowledge about the mechanisms

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governing the protein-manganese interaction. Otherwise, the biomineralization process has

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remained largely uncontrolled, hence hindering its application in areas where time-wise

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particle size and morphology control are required.

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In addition, while previous studies on the Fe-apoferritin system have revealed

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detailed mechanisms of oxidation and mineralization of FeOOH through kinetic analyses,

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the differences between the reaction pathways of the Fe and Mn systems remain largely

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unexplored.27,28,39 Thus, it is unclear whether the mechanism in Fe-apoferritin systems can

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sufficiently explain that in Mn-apoferritin systems. Therefore, the purpose of our study is

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to investigate Mn-apoferritin systems, determining the stoichiometry and elucidating the

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step-wise reaction pathways for Mn oxidation and nucleation processes inside apoferritin

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cavities. In this study, we confirmed the specific phase of Mn(III) (hydr)oxide and provided

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detailed kinetic analyses of MnOOH formation inside apoferritin cavities by quantifying

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solid Mn(III) concentrations with the leucoberbelin blue (LBB) method. We also

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investigated the kinetics of -MnOOH formation. From there, we elucidated the

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similarities and differences in the oxidation mechanisms involved in Fe- and Mn-

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apoferritin systems. Our findings provide a new understanding of the functionality of

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apoferritin for MnOOH synthesis and allow more systematic prediction and control of

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particle sizes and morphology when using macromolecular templates for synthesizing

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metal (hydr)oxide nanomaterials.

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Experimental Section

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Sample preparation

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Sample solutions were prepared using apoferritin extracted from equine spleen (0.2

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µm filtered, Sigma-Aldrich, consisting of around 10% H and 90% L subunits45-47), reagent-

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grade Mn(NO3)2·4H2O (99.98%, Alfa Aesar), AMPSO (99%, Sigma-Aldrich), and aerated

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ultrapure deionized water (resistivity >18.2 MΩ-cm, with 8.4 ± 0.1 mg/L of dissolved O2).

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All samples were prepared in 10 mL 0.05 M AMPSO buffer solution with 0.1 µM

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apoferritin. This preparation allowed us to maintain an initial pH condition during an

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experiment lasting several hours without any changes of the initial concentrations of Mn2+

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(aq) and apoferritin and without precipitation of Mn(OH)2 (s)48, 49, all of which can occur

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if pH is maintained with dynamic titration. The AMPSO buffer has been widely employed

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in previous studies on MnOOH formation inside apoferritin, and thus we consider the use

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of AMPSO to be a proper experimental approach that can be more comparable to those in

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previous literature.43, 44

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To examine the effect of OH- concentration on the rate of MnOOH formation, pH

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conditions of 8.90 ± 0.05, 9.00 ± 0.05, and 9.10 ± 0.05 were investigated. These pH ranges

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were chosen because it enables measuring the nucleation kinetics of MnOOH in apoferritin

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within hours, and can differentiate the nucleation of MnOOH in apoferritin and in solution.

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All samples used for Mn(III) quantification were replicated more than three times to

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account for the high sensitivity of the -MnOOH formation rate in apoferritin to changes

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in pH. To reach the target pH values, proper amounts of 1 M HNO3 solution were added.

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For each pH condition, samples with different Mn2+ concentrations were prepared by

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adjusting the initial Mn2+ atom: apoferritin ratios to be 2000:1, 2500:1, 3000:1, 3500:1, and

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4000:1.

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Samples in test tubes were parafilm-sealed with a small punched hole to allow free

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access to air but to prevent unwanted contamination or significant water evaporation.

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Because oxygen was constantly replenished due to contact with the atmosphere and the

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sample solutions remained equilibrated with oxygen, we assumed that oxygen

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consumption was not a limiting factor for Mn oxidation kinetics. The measured oxygen

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concentration at pH 9.1 with an Mn to apoferritin ratio of 4000:1, which shows the fastest

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oxidation rate among the experimental conditions, also supports considering the oxygen

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concentration as a constant value (Figure S1). To analyze the difference in nucleation

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pathways in systems with and without apoferritin, control experiments were conducted

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without the addition of apoferritin.

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Phase characterization

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The oxidation states of the Mn solid phase in samples prepared with and without

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apoferritin were characterized using X-ray Photoelectron Spectroscopy (XPS,

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PHI 5000 VersaProbe II, Ulvac-PHI with monochromatic Al Kα radiation (1486.6 eV)).

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The Mn 3p spin orbit was used because it provides more accurate information than the Mn

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2p that has less sensitivity to the bonding environment of the mineral.50 Likewise, because

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the intensity of Mn 3s spin orbit was too weak to get information about multiplet splitting,

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we used only Mn 3p spin orbit. From this analysis, the peak positions of experimental

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samples were compared to those of reference samples of Mn(II) and Mn(III). The peak

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positions of Mn(II) and Mn(III) used for reference were 48.1 eV and 48.7 eV, based on the

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average of values reported in previous works and those of purchased samples from the

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natural environment (S2 in the Supporting Information). C 1s at 284.8 eV was used as the

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reference energy level. To determine the oxidation states of Mn, peak positions given by

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the asymmetrical Gaussian−Lorentzian curve-fitting for the experimental samples were

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compared to the reference peak values.

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High resolution transmission electron microscopy (HRTEM, JEOL 2100F) images

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of reaction systems with and without apoferritin were used to observe particle

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morphologies and to identify mineral phases. To identify the phases of Mn(III)

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(oxy)hydroxide nucleated inside apoferritin, samples were first centrifuged at 14,800 rpm

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for 10 minutes to separate potential homogeneous nucleation. The supernatants were

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collected for ultracentrifuging (Thermo Scientific Sorvall WX Ultra Series Centrifuge with

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a T-865 Fixed Angle Rotor) at 40,000 rpm for 30 minutes to obtain concentrated samples

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of Mn-reconstituted ferritin. D-spacing data calculated from the HRTEM-electron

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diffraction (ED) pattern were compared with the literature to further determine the phases

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of reaction products.

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In addition to ED analyses, wide angle X-ray scattering (WAXS) further confirmed

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the mineral phase, formed inside apoferritin cavities. The measurements were conducted

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with an energy of 58.290 keV (λ = 0.2127 Å) on the beamline 11ID-B at the Advanced

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Photon Source at Argonne National Laboratory, IL. A sample reacted for 12 hrs at pH 9.0

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with an Mn to apoferritin ratio of 2000:1 was transferred to a quartz capillary for in situ

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WAXS. The sample was exposed for 180 s. Data from a background sample, prepared

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identically except for adding 200 µM of Mn2+(aq), was also obtained with the same beam

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exposure time for background subtraction.

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Particle size distributions for samples with and without apoferritin were measured

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using dynamic light scattering (DLS, Zetasizer) at different elapsed times. Systems under

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each pH condition were given enough time to react so that the shift in the nucleation

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pathway from inside apoferritin cavities to in solution was completely observed in terms

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of particle size change. The particle size evolutions were monitored for 12, 8, and 5 hr for

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samples prepared at pH 8.9, 9.0, and 9.1, respectively. (S3 in the Supporting Information).

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Colorimetric quantification of Mn(III) (oxy)hydroxide formation in apoferritin

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Leucoberbelin blue (LBB, Sigma Aldrich) is a reducing agent that specifically

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reduces Mn of higher oxidation states to Mn2+, forming a colored species that allows

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colorimetric quantification of the oxidized Mn concentration using UV-vis spectroscopy.51

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LBB solution was prepared by dissolving 0.004 % (w/v) of LBB in de-ionized water, to

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which 45 mM acetic acid was added, and the solution was stored at 4 °C overnight.51 The

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calibration curve was prepared by oxidizing the LBB solution with standard KMnO4

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solutions, with the extinction coefficient, ɛ, determined to be 205,000 M-1 at a wavelength

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of 625 nm.

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To prepare the samples for colorimetric measurement, an aliquot of 0.3 mL was

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taken from the reacting solutions and 1.5 mL of LBB solution was added. The samples

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were allowed to equilibrate in the dark for at least 30 minutes, after which the absorbance

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at 625 nm was measured using UV-Vis spectroscope (UV-Vis, Cary 50 Bio UV-Vis

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spectroscopy, Varian Inc.,).

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To understand how LBB molecules gain access to Mn(III) mineral cores, we

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analyzed the apoferritin particle size and morphology at varied pH conditions using atomic

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force microscopy (AFM, Nanoscope V multimode SPM, Veeco Inc.) in tapping mode.

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TEM images of samples after LBB treatment were also obtained to support the validity of

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using the LBB method for quantifying heterogeneously nucleated Mn(III) inside the

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apoferritin cavity. To visualize the protein, the TEM samples were negatively stained with

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uranyl acetate (Electron Microscopy Sciences) and washed thoroughly with DI water.

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Kinetic analyses

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Equation (1) was hypothesized to depict the formation kinetics of -MnOOH,

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which we identified to be the phase of the reaction product formed inside apoferritin (S4

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in the Supporting Information).

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= k[Mn2+]a [OH-]b

(1)

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The LBB-probed -MnOOH concentrations over time were then fitted linearly.

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The rate of Mn(III) formation at each experimental condition was then calculated from the

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slope of the linear regression lines. Non-linear regression fittings then provided the best-

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fit values of the overall rate constant for the combined oxidation and nucleation of

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α-MnOOH and the orders of reaction with respect to concentrations of Mn2+ and OH-.

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Based on the kinetic parameters obtained, we suggested the best predicted stoichiometric

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numbers of reacting species (a and b in Equation (1)) and mechanism for the chemical

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reactions involved. The proposed mechanism was further confirmed from the detection of

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low concentrations of H2O2 by the peroxidase-catalyzed n,n-diethyl-p-phenylenediamine

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oxidation method (S4 in the Supporting Information).

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Results and Discussion

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Identification of -MnOOH nanoparticle formation inside the apoferritin cavities

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Using XPS and TEM analyses, we identified two distinct nucleation pathways of

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-MnOOH nanoparticle formation inside apoferritin cavities, as well as and larger-sized

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Mn(OH)2 (s) particles (with an oxidized surface layer of Mn3O4) formed in bulk solution

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not associated with apoferritin. The oxidation states of products formed from reactions in

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the presence of apoferritin were measured using XPS (Figure 1a). The samples prepared

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with apoferritin had a Mn 3p peak position at 48.6 eV, which is in accord with the average

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of Mn(III) values reported in the literature and with our own reference samples (48.7 eV)

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(Table S1).

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To further characterize the phases of nucleated products inside apoferritin cavities,

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TEM images together with electron diffraction (ED) patterns were obtained for the sample

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prepared at pH 9 and with 200 µM Mn2+ (Figure 1b). Because Mn(OH)2 (s) forms more

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readily with an elevated Mn2+ concentration in solution, the lowest Mn(II)/apoferritin ratio

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(2000:1) was chosen for TEM analyses of nucleation inside apoferritin to avoid possible

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confusion from Mn(OH)2 (s) formed in solution. D-spacings of the solid phase were

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subsequently calculated from the ED pattern and compared with reference values of

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manganite (-MnOOH), groutite (α-MnOOH), feitknechtite (-MnOOH), pyrochroite

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(Mn(OH)2 (s)), and hausmannite (Mn3O4). Although both groutite and manganite have a

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d-spacing value of around 2.67 Å, as observed by TEM (Figure 1b), ED analyses clearly

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showed two large d-spacings of 5.14 and 4.45 Å, which match only with the reference

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d-spacings of groutite (α-MnOOH) faces of (200) and (101). In addition to ED analyses,

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from in situ measurements of synchrotron-based wide angle X-ray scattering (WAXS)

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(Figure 1c), we also clearly observed the strong diffraction pattern of α-MnOOH at 21°,

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which is the strongest diffraction of α-MnOOH. The WAXS results further support the ED

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analyses by confirming the phase of the nucleation product inside apoferritin cavities to be

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α-MnOOH. Thus, we can rule out possible effects of ultracentrifugation and drying on the

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phase transformation of synthesized α-MnOOH nanoparticles in apoferritin. Our finding

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differs from that provided by Mann’s group, who found that the phase of ferritin-

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encapsulated Mn resembles - and -MnOOH, based on X-ray absorption spectroscopy

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(XAS).44 The difference in mineral phase formation between our study and Mann’s study

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could arise because of varied experimental conditions. The concentration of apoferritin

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(Mann’s study used 2.25 µM, while we used a much lower concentration, 0.10 µM) could

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affect the phases of final reaction products due to different saturation conditions with

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respect to the potential Mn(III) (oxy)hydroxide phases.44

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To complement what we found for solid phase Mn(III) formation inside apoferritin

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cavities, the phase of Mn (hydr)oxide particles formed in solution (from samples prepared

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without apoferritin) were identified by XPS, XRD, and TEM (Figure 1a and Figure S5).

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The XPS spectrum shows the peak position for a sample prepared without apoferritin is at

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48.2 eV, which is in between the reference values of Mn(II) (48.1 eV), and Mn(III) (48.7

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eV), indicating the formation of a mineral phase different from that found in the sample

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prepared with apoferritin (i.e., a peak shown at 48.6 eV). Further characterization by XRD

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and TEM confirms that the phase of nucleation in the bulk solution is mainly pyrochroite

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(Mn(OH)2 (s)) with a small hint of oxidized hausmannite (Mn3O4) on the surface, with a

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much bigger particle size (> 50 nm) (Figure S5b) than that of nanoparticles nucleated in

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apoferritin (~8 nm) (Figure 1b). Comparison between nucleation in solution and that within

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apoferritin cavities suggests that apoferritin affects Mn oxidation and nucleation by altering

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the phase of the reaction product and controlling the particle size.

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To monitor the evolution of the dominant nucleation pathway in Mn-apoferritin

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systems, from inside apoferritin cavities to in the bulk solution, we measured the particle

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size distributions of samples prepared under varied experimental conditions (pH 8.9–9.1

253

and Mn(II)/protein ratios 2000:1–4000:1) at different elapsed times. Representative

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particle size distributions under pH 8.9, 9.0, and 9.1 with Mn(II)/protein ratios 3500:1 are

255

shown in Figure 2 and the complete data set is available in Figure S3. The sizes of the

256

particles remained around 11 nm at the early stage of reaction (before 8, 6, and 4 hr,

257

respectively for samples prepared under pH 8.9, 9.0, and 9.1), indicating that the particles

258

formed mainly inside the apoferritin cavity, which has an outer diameter of 12 nm.19 As

259

the reaction continues, sharp increase in particle size suggests nucleation in solution

260

dominates over apoferritin in number concentration. Together with XPS and TEM

261

characterizations, the DLS data suggested there are two distinct nucleation pathways: one

262

inside the apoferritin and another in the bulk solution (Figure S3 and Figure S6). Mn

263

oxidation and mineralization inside apoferritin cavities forms α-MnOOH nanoparticles

264

with sizes confined to the dimension of the cavity (Figure S3). Nucleation in the bulk

265

solution forms combined pyrochroite (Mn(OH)2 (s)) and hausmannite (Mn3O4) with larger

266

sizes (> 50 nm) upon oxidation by dissolved oxygen (Figure S5 and Figure S6). The shift

267

in particle size profiles for samples prepared under varied experimental conditions also

268

indicate that nucleation inside apoferritin cavities is preferred to that in solution at the early

269

period of reaction (before 8, 6, and 4 hr respectively for samples prepared under pH 8.9,

270

9.0, and 9.1). The thermodynamics suggests that the preference could be due to the reduced

12


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271

energy barrier provided by iron-coordinating amino acid residues (His65, Glu27, Glu61,

272

Glu62, and Glu 107 for ferritins) at the ferroxidase center that catalyzes Mn oxidation,

273

similar to the reaction pathway in the protein catalysis mechanism reported in iron

274

deposition studies.52 As the nucleation sites in apoferritin approach saturation, nucleation

275

formed in bulk solution dominates as the reaction proceeds. We also found that the

276

transition between the two nucleation pathways is highly dependent on the initial

277

experimental conditions (Figure S3). The transition from nucleation inside apoferritin to

278

nucleation in bulk solution takes place earlier at higher pH and with higher Mn2+

279

concentration, implying that OH- and Mn2+ are rate-determining agents for apoferritin-

280

mediated heterogeneous oxidation and nucleation.

281

LBB quantification of -MnOOH nanoparticles formed inside apoferritin cavities

282

To obtain the combined rate of both oxidation and nucleation of Mn inside

283

apoferritin, α-MnOOH concentrations inside apoferritin cavities were quantified

284

colorimetrically by LBB, which is a reducing agent that specifically reduces Mn of higher

285

oxidation states to Mn2+. The LBB molecule, with its multiple benzene-ring structure, is

286

not likely to enter the protein’s interior through the hydrophilic channels because the

287

narrowest part of the channels is only 3 to 4 Å across.22 Previous research by Kim et al.

288

discovered that the apoferritin structure will undergo gradual disintegration under pH

289

below 3.40.47 Thus, we hypothesized that LBB penetrates the protein shell through the

290

disintegrated protein and hence reduces the α-MnOOH core. To test this hypothesis, the

291

particle size profiles of apoferritin solutions under pH 9.0, 3.0, and 1.0 were captured with

292

DLS. The large z-average (the intensity weighted mean hydrodynamic size) for pH 9.0

293

might result from the presence of impurities. Despite the interference from the background

13



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294

condition, as shown in Figure 3d, the z-average of the apoferritin samples increases

295

significantly with lower pH, which confirms that aggregation occurred as regular structures

296

of apoferritin were disrupted. AFM images confirmed the effects of pH on apoferritin

297

morphologies (Figure 3a–c). At pH 9.0, apoferritins generally retain the well-defined

298

spherical shape, suggesting intact structures. As the pH was lowered to 3.0, the particle

299

morphologies became less identifiable, and aggregations of disassembled apoferritin

300

fragments appear in the AFM images. At pH 1.0, the tertiary structures of the proteins were

301

completely lost, and an elongated conformation of unfolded polypeptides was observed.

302

The pH of LBB solution used in our study was 3.1. Based on the AFM and DLS results,

303

we concluded that under the low pH condition induced by the LBB solution, LBB

304

molecules are able to traverse the protein shell and reduce the α-MnOOH core in the central

305

cavity through fragmented surfaces resulting from the acidic pH environment.

306

To further confirm the successful reduction of the α-MnOOH by the LBB method,

307

TEM images of samples after LBB treatment were obtained (Figure 3e). The negative

308

staining shows protein aggregation in post-LBB treated samples, again confirming the

309

deterioration of apoferritin in the LBB solution. Also, we detected d-spacing values of 3.30

310

Å, which are consistent with the reference values of uranyl acetate (Figure 3e), while no

311

values that could possibly match with Mn (hydr)oxide phases were identified. The absence

312

of solid phase Mn(III) in TEM images (Figure 3e) suggests that the α-MnOOH particles

313

formed inside apoferritin cavity have been completely reduced to Mn2+, and thus the LBB

314

method provides a reliable means for quantifying α-MnOOH deposition inside apoferritin

315

cavities.

14


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316

Determination of the combined rates of oxidation and nucleation of Mn inside

317

apoferritin from pseudo-zeroth order kinetics

318

To determine the combined rate of Mn oxidation and nucleation inside apoferritin using

319

the LBB method, we measured the concentrations of Mn(III) in samples prepared under all

320

experimental conditions at different elapsed times. The combined oxidation and nucleation

321

process inside apoferritin obeyed pseudo-zeroth order kinetics, with Mn2+ and OH- being

322

the rate-determining agents. As shown in Figure S3, particle size profiles of the samples

323

indicated that the transition of nucleation from inside apoferritin to in the bulk solution

324

depends on both pH and Mn2+ concentrations. Therefore, to obtain accurate kinetics solely

325

for nucleation occurring inside apoferritin cavities, we restricted the duration of the

326

reaction to before the transition, so that effect of Mn(OH)2 (s) formation in the bulk solution

327

was not taken into account. For pH 8.9 and 9.0, concentrations of Mn(III) were measured

328

at 2 hr intervals to 8 hr; for pH 9.1, concentrations of Mn(III) were measured at 0.5 hr

329

intervals to 3 hr because of the markedly more rapid MnOOH formation from

330

heterogeneous nucleation at the slightly higher pH. The saturation indices of Mn(OH)2 (s)

331

were calculated to explain the more rapid nucleation in solution with increasing pH and

332

Mn2+ concentrations (Table S2 and S3 in the Supporting Information). α-MnOOH

333

concentration profiles for each experimental condition are shown in Figure 4. A linear

334

increase in Mn(III) concentrations with time is seen and can be explained from the pseudo-

335

zeroth order kinetics. Because unlimited access to air was guaranteed for all samples during

336

reaction, and because oxygen consumption by Mn oxidation was negligible compared to

337

the total dissolved oxygen in the solution, the oxygen level was assumed to remain

338

unchanged throughout the reaction. Also, the Mn concentration was assumed to be constant

15



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339

due to the high Mn atom to apoferritin ratio (higher than 1000:1) used in this study.

340

Therefore, we fitted the experimental data with linear regression lines and obtained the

341

initial rate of reaction by calculating the slope of each (Figure 4). The initial rate of reaction

342

increases with pH and Mn2+ concentrations, which confirms that the OH- and Mn2+ are

343

rate-determining agents.

344

Overall reaction rates and the proposed mechanism

345

By fitting the overall rates of oxidation and nucleation obtained from experiments

346

to the hypothesized rate equation, Equation (1), we specified the kinetic parameters in the

347

rate equation and proposed that -MnOOH nanoparticles form in sequential steps of

348

oxidation and mineralization at the ferroxidase center of apoferritin with the involvement

349

of two intermediates. To obtain the rate constant, k, and the orders of reactions with regard

350

to OH- and Mn2+, a and b as specified in Equation (1), we fitted the experimentally obtained

351

rates of reaction to Equation (1) with assumed combinations of a and b, using non-linear

352

least squares regression (Figure 5). The rate equation best describes the experimental data

353

when a and b are 2 and 4, respectively, under which case the best-fit value of the overall

354

rate constant, k, is 1.28  10-9 M-5hr-1 (Figure 5). The high order of reaction with respect

355

to OH- also explains the high sensitivity of the reaction systems to even minor changes in

356

pH condition (Figure 5b). To further support the validity of the fitting results, we analyzed

357

the goodness of fit by calculating R squares for a range of a and b values. The best-fit

358

results give a R square value of 0.9953, confirming the high accuracy with which the model

359

describes the experimental data (S7 in the Supporting Information).

360 361

Based on the kinetics parameters predicted by the computational fitting, we propose here a possible mechanism for enzymatically-catalyzed Mn(III)OOH formation.

16


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362

Oxidation: 2Mn2+ + O2 + 4OH- + P

P-[MnIII2O2(OH)2] + H2O2

(2)

363

Disassociation: P-[MnIII2O2(OH)2]

[MnIII2O2(OH)2] + P

(3)

364

Mineralization: [MnIII2O2(OH)2]

365

Overall: 2Mn2+ + O2+ 4OH- → 2MnIIIOOH(core) + H2O2

2MnIIIOOH(core)

(4) (5)

366

Here, P is the protein, k1, k2, and k3 are the rate constants for the respective

367

elementary steps. We propose that sequential oxidation and mineralization reactions with

368

the formation of two intermediates lead to the overall α-MnOOH deposition in apoferritin.

369

The rate-determining step in the oxidation process generates the Mn(III)-apoferritin

370

complex, which forms at the ferroxidase dinuclear center because of protein catalysis. The

371

oxidized Mn(III) then rapidly dissociate from the binding sites in the protein shell. The free

372

Mn(III) subsequently diffuses into the protein cavities, where mineralization occurs to form

373

nascent -MnOOH core. The formation of intermediates in the Mn-apo system can be

374

justified from the iron deposition process in native ferritin. Previous studies reported that

375

the first intermediate formed from Fe(II) oxidation at the ferroxidase center by oxygen is

376

μ-1,2-peroxodiFe(III).26, 53-56 The initial intermediate subsequently dissociates to one or

377

more μ-oxo(hydroxo)-bridged diFe(III) intermediate(s), which ultimately lead to the

378

formation of the nascent mineral core. Based on the Fe studies, enzymatically catalyzed

379

oxidation reactions occurring at the ferroxidase dinuclear center might also exist for the

380

Mn-apoferritin system and explain the observed kinetics in this study, although the phase

381

and structure of the intermediates formed in the two systems differ from each other because

382

of the difference in the reaction stoichiometry.

383 384

In this study, by assuming pseudo-steady state kinetics for P-[Mn2O2(OH)2], we derived the following expression for P-[Mn2O2(OH)2]: 17



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385

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P-[Mn2O2(OH)2] =k1/k2[O2][Mn2+]2[OH-]4.

(6)

386

Next, by assuming pseudo-steady state kinetics for [Mn2O2(OH)2] and substitute Equation

387

(6) into the expression, we obtained Equation (7) for [Mn2O2(OH)2]

388

[Mn2O2(OH)2] = k2/k3 P-[Mn2O2(OH)2] = k1/k3[O2][Mn2+]2[OH-]4.

389

(7)

390

By substituting Equation (7) into the rate equation for α-MnOOH formation (Equation (1)),

391

we obtained Equation (8)

392

= k1[O2][Mn2+]2[OH-]4.

(8)

393

The product of the elementary rate constant, k1 and dissolved oxygen concentration can be

394

lumped into the overall rate constant, k, and the rate equation can be rearranged into the

395

final form

396

= k[Mn2+]2[OH-]4.

(9)

397

Because the overall reaction forms H2O2 as one of the final products (Equation (4)),

398

to further test the validity of the proposed reaction stoichiometry, we used the peroxidase-

399

catalyzed n,n-diethyl-p-phenylenediamine oxidation method to detect the presence of H2O2

400

(S4 in the Supporting Information). The UV spectra obtained after 4 hr and 12 hr of reaction

401

show clear absorbance peaks at 551 nm.57 Because of the instability of H2O2, we focused

402

on confirming the presence of H2O2 rather than obtaining the exact H2O2 concentration.

403

The presence of strong peak at 551 nm (Figure S4) confirmed the formation of H2O2 during

18


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404

the overall reaction. Based on the intermediate formation and the presence of H2O2, we

405

suggest that separate oxidation and mineralization reactions are involved in the overall

406

MnOOH formation in apoferritin.

407

To extend our discussion of the reaction stoichiometry and the catalytic function of

408

apoferritin on α-MnOOH formation to the synthetic processes of other nanomaterials, we

409

compared the deposition of Mn in apoferritin to that of Fe. As shown in Equation (5), the

410

overall stoichiometry involved in α-MnOOH formation is similar to the protein catalysis

411

mechanism proposed in Fe studies in the respect that both systems undergo apoferritin-

412

mediated oxidation reactions.29, 58 The similarity suggests that Mn2+ up-taken into the

413

protein is likely to go through the ferroxidase center, where a series of key glutamate

414

residues on the exposed surface serve to catalyze the oxidation of Mn.20, 29, 58 However, the

415

deposition of Mn in apoferritin is distinguished from Fe(III) oxy(hydr)oxide formation in

416

that the order of reactions with respect to the metal and hydroxyl are 2 and 2, respectively

417

in the protein catalysis model proposed in the Fe study, while we found the orders were 2

418

and 4 for the Mn-apoferritin system. This difference in kinetic aspect suggests that despite

419

protein catalyzes the oxidation reactions in both cases, the reactions themselves are

420

different, forming intermediates of different phases and structures. In addition, the protein

421

catalysis mechanism is applicable for even high Mn(II)/apoferritin ratios (higher than 1000

422

:1). In the Fe case, the mechanism occurs only transiently at the incipient stage of reaction

423

under low Fe flux (less than 48 Fe(II)/protein), and reactions occur directly on the mineral

424

surface without protein catalysis as Fe flux is increased above the threshold ratio.29, 58 The

425

slower oxidation kinetics of Mn might have contributed to this difference in enzymatic

426

function of apoferritin in Mn and Fe systems. Despite the difference in phases of

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

427

intermediates and kinetics, this mechanistic study also elucidates the role of the ferroxidase

428

center shared between the two systems, suggesting that the enzymatic sites of the protein

429

may engage in the formation of other metal-oxyhydroxide(s) in similar ways. Thus, this

430

work highlights the importance of kinetic control in mechanistic studies or nanomaterial

431

synthesis when using apoferritin as the mineralization platform. This new information can

432

benefit in the optimization of bio-inspired synthesis of uniform-sized nanomaterials.

433

Conclusions

434

We investigated the kinetics and mechanism of Mn oxidation and nucleation in

435

apoferritin. We identified two distinct nucleation pathways in an apoferritin-Mn system:

436

nucleation inside apoferritin cavities forms α-MnOOH nanoparticles with sizes of 6–8 nm,

437

and nucleation in solution forms Mn(OH)2 (s) (with an oxidized layer of Mn3O4) particles

438

of larger sizes (> 50 nm). Nucleation of α-MnOOH in apoferritin is favored at the early

439

stage of reaction, then nucleation of Mn (hydr)oxides in solution gradually dominates as

440

the reaction continues. We confirmed that the step-wise disassembly of apoferritin under

441

acidic conditions induced by LBB solution allows the LBB molecules to traverse the

442

protein shell, and thus quantify the nucleated α-MnOOH in apoferritin as a complete

443

reduction of Mn(III). From the fitting results, we found that Mn2+ and OH- are rate-

444

determining agents with best-fit orders of reaction of 2 and 4, respectively. Based on kinetic

445

analyses, we proposed that α-MnOOH forms along with H2O2 through sequential steps of

446

oxidation and mineralization, with the possible involvement of two intermediates. Our

447

findings illustrate the appropriate experimental conditions that allow pure synthesis of α-

448

MnOOH nanoparticles in apoferritin without being affected by unwanted Mn (hydr)oxide

449

nucleation in solution. In addition, the suggested oxidation and nucleation mechanisms 20


Page 22 of 34

Page 23 of 34

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450

will provide implications to understand the formation behaviors of other inorganic

451

nanoparticles using other protein cages as macromolecular templates through

452

biomineralization.

453

Associated Contents

454

Supporting Information. Dissolved oxygen (S1), XPS references (S2), particle size

455

profiles for samples with 0.1 M apoferritin solution and saturation indices (S3), N,N-

456

diethyl-p-phenylenediamine (DPD) method for detection of H2O2 (S4), phase

457

identification of nucleation of Mn (hydro)oxides in solution (S5), particle size profiles for

458

samples without apoferritin solution (S6), and evaluation of fitting results (S7). The

459

Supporting Information is available free of charge on the ACS Publications website.

460

Author Information

461

Corresponding Author

462

*E-mail: [email protected]

463

Author Contributions

464

The manuscript was written through contributions of all authors. All authors have given

465

approval to the final version of the manuscript.

466

†

467

Notes

468

The authors declare no competing financial interests.

469

Acknowledgments

Y. H. and H. J. contributed equally.

470

The authors would like to acknowledge the support from the National Science

471

Foundation’s Environmental Chemical Sciences program (CHE-1610728). We also thank 21



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472

Washington University’s Institute of Materials Science & Engineering (IMSE) for use of

473

XPS and TEM, and Professor James C. Ballard for carefully reviewing the manuscript.

474

Work at the Advanced Photon Source (Sector 11 ID-B) at Argonne National Laboratory

475

was supported by the US Department of Energy, Office of Science, Office of Basic Energy

476

Sciences, under Contract No. DE-AC02-06CH11357.

22


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List of Figures Figure 1. (a) XPS spectra for samples prepared at pH 9.0 and with 200 µM Mn2+, with and without apoferritin. (b) TEM image and ED pattern of nucleated Mn(III) inside apoferritin prepared at pH 9.0 and with 200 µM Mn2+. (c) The background subtracted results of the in situ measurement of the synchrotron-based wide angle X-ray scattering used in identifying the phase of solid-state Mn(III) formation inside apoferritin cavities Figure 2. DLS particle size profiles for samples prepared under (a) pH 8.9, (b) pH 9.0, and (c) pH 9.1 with 350 M Mn2+. Figure 3.

AFM images of 0.1M apoferritin solution prepared under (a) pH 9, (b) pH 3,

and (c) pH 1. Height profiles were obtained at white-dotted lines. (d) Z-averages (intensity weighted mean hydrodynamic sizes) from DLS measurement for 0.1M apoferritin solution prepared under pH 9.0, 3.0, and 1.0. (e) TEM image of post-LBB treated sample prepared under pH 9.0 and with 200 M Mn2+, after negative staining with uranyl acetate. Figure 4. Experimentally measured concentrations of Mn(III) determined using the LBB method, and fitted linear regression lines for samples with different Mn(II)/protein ratios at (a) pH 8.9, (b) pH 9.0, and (c) pH 9.1. Figure 5.

Rates of overall Mn oxidation and nucleation inside apoferritin cavities

determined from the slopes of linear regression lines in Fig. 3 and fitted results using Equation (1), with a = 2, b = 4, and k = 1.28  10-9 M-5hr-1 with respect to (a) Mn2+ concentration and (b) pH.

26


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Figure 1

27



3 hr 6 hr 8 hr 12 hr

40 30

Hetero. 20 Nucleation (< 8hr)

Homo. Nucleation (> 8hr)

10 0

1

10

100

Particle Size (nm)

1000

(c)

pH 9.0 2 hr 4 hr 6 hr 8 hr 10 hr

40 30 20


Hetero. Nucleation (< 8hr)

10 0

1

10

100

Number Percentage (%)

(b)

pH 8.9

Number Percentage (%)

(a) Number Percentage (%)

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1000

Particle Size (nm)

Figure 2

28


pH 9.1 1 hr 2 hr 3 hr 4 hr 5 hr

40 30 20


Hetero. Nucleation (< 4hr)

10 0

1

10

100

Particle Size (nm)

1000

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Figure 3

29



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Figure 4

30


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(a)

6

pH 8.9 pH 9.0 pH 9.1

4 2 0

200

Mn

(b)

Rate of Reaction (M/hr)

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Reaction rate (M/hr)

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6 4

2+

300

400 Concentration (M)

2000:1 2500:1 3000:1 3500:1 4000:1

2 0

8.9

9.0

pH

Figure 5

31


9.1


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Table of Contents Graphic and Synopsis (For Table of Contents Use Only)

Synopsis: This work investigated the kinetics of -MnOOH nanoparticle formation inside apoferritin cavities and proposed mechanisms for the Mn oxidation and nucleation reactions. Title: Kinetics of α-MnOOH Nanoparticle Formation through Enzymatically-catalyzed Biomineralization inside Apoferritin. Authors: Yue Hui†, Haesung Jung†, Kim Doyoon, and Young-Shin Jun*. †

These authors contributed equally *To whom correspondence should be addressed

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Kinetics of Î±-MnOOH Nanoparticle Formation through Enzymatically

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