Communication pubs.acs.org/IC
Mimicking a Natural Enzyme System: Cytochrome c Oxidase-Like Activity of Cu2O Nanoparticles by Receiving Electrons from Cytochrome c Ming Chen,† Zhonghua Wang,*,† Jinxia Shu,† Xiaohui Jiang,† Wei Wang,† Zhen-Hua Shi,‡ and Ying-Wu Lin*,‡ †
Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637002, P. R. China ‡ School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, P. R. China S Supporting Information *
and many nanomaterials have been found to possess enzyme-like activities, such as peroxidase-like and oxidase-like activities.6,19−23 Despite this progress, the substrates investigated so far have been limited to small organic molecules, such as 3,3′,5,5′-tetramethylbenzidine, 18,22,24,25 o-phenylenediamine,22,26 diazoaminobenzene,26 etc. Moreover, it has not been attempted to use nanomaterials to mimic the natural enzyme systems because no biomacromolecule has been found to be a substrate or partner for nanomaterials. In this study, we found that cuprous oxide nanoparticles (Cu2O NPs) can receive electrons from ferrous Cyt c and possess CcO-like activity, which thus closely mimics the natural Cyt c−CcO enzyme system. Moreover, the CcO-like activity of Cu2O NPs was shown to be dependent on the O2, pH, and Cu2O size. We first prepared Cu2O NPs according to a reported method27 (see the Supporting Information, SI, for details). The transmission electron microscopy (TEM) image showed that the morphology of the as-prepared Cu2O NPs was cubic-like with an average size of 50 nm (Figure 1a and inset), which agrees well with the literature.27 X-ray diffraction (XRD) of the Cu2O NPs was shown in Figure 1b, in which all diffraction peaks were well indexed to the standard cubic phase of the Cu2O crystal with a lattice constant of a = 0.427 nm (JCPDS 05-0667). The diffraction peaks at 2θ of 29.4°, 36.3°, 42.1°, 61.3°, 73.5°, and 77.3° can be ascribed to the reflections of the (110), (111), (200), (220), (311), and (222) planes of cubic Cu2O, respectively. We further performed X-ray photoelectron spectroscopy (XPS) analysis to investigate the surface composition of the Cu2O NPs. As shown in Figure 1c for the high-resolution XPS spectrum of Cu 2p, the binding energies for Cu 2p1/2 and Cu 2p3/2 were observed at 952.3 and 932.3 eV, respectively, in good agreement with the reported binding energies for Cu2O.22,28,29 The O 1s XPS spectrum was shown in Figure 1d, with a broad band at ca. 531 eV. After nonlinear least-squares fitting using a Lorentzian−Gaussian model, the broad O 1s peak was divided into three peaks. The peak at a lower-energy value of 530.28 eV was attributed to the surface bridging oxygen of Cu2O.30,31 The peak at a medium value of 531.48 eV was ascribed to the surface hydroxyl oxygen,32,33 and the peak at a higher binding energy of
ABSTRACT: Inorganic nanomaterials-based artificial enzymes (nanozymes) have received considerable attention over the past years. However, the substrates studied for nanozymes have so far been limited to small organic molecules. The catalytic oxidation of biomacromolecules, such as proteins, by nanozymes has not yet been reported to date. In this study, we report that cuprous oxide nanoparticles (Cu2O NPs) possess cytochrome c oxidase (CcO)-like activity and catalyze the oxidation of cytochrome c (Cyt c), converting it from the ferrous state to the ferric state under atmospheric oxygen conditions. Furthermore, the CcO-like activity of Cu2O NPs is pH- and size-dependent. The lower the solution pH and the smaller the particle size, the higher the CcO-like activity. The artificial Cyt c−Cu2O NPs system closely mimics the native Cyt c−CcO enzyme system, which opens new vistas in enzyme construction and potential applications.
N
atural enzymes have been extensively studied because of their high substrate specificity and high catalytic efficiency under mild conditions. However, they bear some intrinsic disadvantages, such as high cost of preparation and low stability under extreme conditions.1,2 Therefore, attempts to construct enzyme mimics as highly stable and low-cost alternatives to natural enzymes have been increasing for chemists and biochemists, with remarkable progress such as extensive investigations for cyclodextrins, metal complexes, polymers, and biomolecules.3−12 In addition, natural enzymes usually perform biological functions effectively by forming complexes with their partners. For example, cytochrome c oxidase (CcO) forms a complex with cytochrome c (Cyt c) by receiving electrons, as is required for the four-electron reduction of oxygen to water at its heme−copper dinuclear center (O2 + 4H+ + 4e− → 2H2O).13 Meanwhile, it is a challenge to mimic the natural enzyme systems, and little progress has been made in this field. Nanomaterials have been favored and widely used as catalysts in the past few decades.14−17 However, the enzyme-like activity of nanomaterials was not discovered for a long time until the report that Fe3O4 nanoparticles possess intrinsic peroxidase-like activity.18 Since then, much attention has been paid to this field, © 2017 American Chemical Society
Received: June 8, 2017 Published: July 28, 2017 9400
DOI: 10.1021/acs.inorgchem.7b01393 Inorg. Chem. 2017, 56, 9400−9403
Communication
Inorganic Chemistry
The oxidation state change was also confirmed by circular dichroism (CD) spectra in the 250−600 nm region (Figure S2). Assuming that the oxidation of Cyt c obeys pseudo-first-order kinetics (−dC/dt = kC), the pseudo-first-order rate constant can be deduced by the plots of ln(C/C0) versus reaction time, where C and C0 are the concentrations of reduced Cyt c at time t and the beginning of oxidation (t = 0). The rate constant was calculated to be 0.0384 min−1 in the presence of Cu2O NPs, which was ∼19fold higher than that of 0.00201 min−1 for the autooxidation of ferrous Cyt c in the absence of Cu2O NPs (Figure S3). These observations indicated that Cu2O NPs efficiently receive electrons from Cyt c by catalyzing its oxidation. To rule out the possibility that the observed Cyt c oxidation was catalyzed by copper ions leached from Cu2O NPs, a leaching solution was prepared by dispersing Cu2O NPs into a phosphate buffer (pH 7.2) with 5 min of sonication. After further incubation for 1 h, the Cu2O NPs were removed by centrifugation. We performed the Cyt c oxidation reaction using a leaching solution instead of Cu2O NPs under the same conditions (see the SI for details). The results showed that the leaching solution exhibited much lower activity compared with Cu2O NPs, and ca. 24% of reduced Cyt c was oxidized after 60 min (Figure 2b), with a pseudo-first-order rate constant (0.00457 min−1; Figure S3) ∼8fold lower than that with Cu2O NPs (0.0384 min−1). Meanwhile, the oxidation rate of reduced Cyt c using a leaching buffer was slightly faster than that with no addition of Cu2O NPs, which might be due to the presence of a small amount of residual Cu2O NPs in the solution after centrifugation. In addition, to rule out the possibility that the ferrous Cyt c was directly oxidized by Cu2O, we carried out XRD measurements. This showed that the crystal phase of the Cu2O sample was not changed after the catalytic oxidation of ferrous Cyt c. The diffraction peaks were still well-matched with cubic Cu2O, and no Cu diffractions were observed in the XRD patterns (Figure 1b). Moreover, the morphology and valence states of Cu2O NPs did not change after the reaction (Figure S4). Electron paramagnetic resonance (EPR) further showed that the trace Cu2+ ions in Cu2O NPs did not change upon reaction with ferrous Cyt c (Figure S5). These results thus confirmed that Cu2O was not reduced to Cu or oxidized to Cu2+; instead, it presumably acted as a CcO-like enzyme. To probe the role of O2 in the Cyt c oxidation reaction, the oxidation of ferrous Cyt c catalyzed by Cu2O NPs was performed with a N2-saturated buffer under a nitrogen atmosphere. The results showed that, although spectral changes were observed, the rate constant in a N2-saturated solution was determined to be 0.00938 min−1 (Figure S6), which was much lower than that in an air-saturated buffer (0.0384 min−1). This observation suggested that the O2 dissolved in the solution was presumably involved in the oxidation of Cyt c, as in a native Cyt c−CcO system. Moreover, the O2 adsorbed on the surface of Cu2O NPs may also be involved in the reaction (Figure 1d).19 Previous studies have also shown that O2 can be adsorbed on the surface of Cu2O and other metal oxides.19,29,31 Therefore, both sources of O2, dissolved in the solution and adsorbed on the surface of Cu2O NPs, were found to be involved in Cyt c oxidation. Fluorescence and EPR spin-trapping studies further showed no formation of reactive oxygen species such as H2O2, ·O2−, or ·OH radicals (Figures S7 and S8), which indicated that O2 was presumably reduced to water as catalyzed by Cu2O NPs by receiving electrons from ferrous Cyt c. To further confirm that O2 participated in the Cyt c oxidation, we first carried out the reaction in a N2-saturated buffer under a
Figure 1. (a) TEM image of the Cu2O NPs. Inset: Size distribution histogram of Cu2O NPs. (b) Powder XRD pattern of Cu2O NPs. (c) Cu 2p and (d) O 1s high-resolution XPS spectra of Cu2O NPs. The standard XRD pattern of cubic Cu2O (JCPDS 05-0667) is also shown in part b for reference.
532.88 eV was probably associated with adsorbed O2 on the surface of Cu2O NPs.29 Note that the adsorption of small molecules and ions (such as water, hydroxyl, and oxygen) on the surfaces of other metal oxides was also observed previously.19,21,28 To examine whether Cu2O NPs exhibit any enzyme-like activity toward biomacromolecule, we chose Cyt c as a substrate. Because in a natural Cyt c−CcO enzyme system Cyt c provides electrons to CcO where O2 is reduced to water, we monitored the electron-transfer reaction between ferrous Cyt c and O2 in the presence of Cu2O NPs by kinetic UV−vis spectroscopy. Upon the addition of Cu2O NPs, the Soret band (414 nm) of the ferrous Cyt c decreased and blue-shifted to 409 nm, and concurrently, the α band (550 nm) gradually decreased with time (Figure 2a). Along with the spectral changes, the color of the Cyt
Figure 2. (a) UV−vis spectral changes in the oxidation of reduced Cyt c by O2 in the presence of Cu2O NPs. Inset: Color change of ferrous Cyt c upon oxidation. (b) Kinetic traces showing the decrease of reduced Cyt c (following the intensity at 550 nm) in the absence or presence of Cu2O and in the presence of a leaching solution.
c solution also changed from pink to yellowish red (Figure 2a, inset), indicating the conversion of ferrous Cyt c to the ferric form.34 After 60 min, more than 90% of reduced Cyt c was oxidized to its ferric state in the presence of Cu2O NPs, with the turnover numbers (i.e., the number of Cyt c molecules reduced per Cu2O NP or per Cu2O unit cell on the surface) estimated to be 5 × 106 and 6.2, respectively (see the SI for calculations), whereas only 12% was oxidized in the absence of Cu2O NPs (Figure 2b). Similar changes were observed by the continuous addition of reduced Cyt c to the reaction solution (Figure S1). 9401
DOI: 10.1021/acs.inorgchem.7b01393 Inorg. Chem. 2017, 56, 9400−9403
Communication
Inorganic Chemistry
the oxidation of ferrous Cyt c increased with an increase of [H+] from 10−8 to 10−6 mol·L−1 (i.e., the pH decreased from 8.0 to 6.0). The rate constant at pH 6.0 was calculated to be 0.0490 min−1, which is ∼3-fold higher than that at pH 8.0 (0.0163 min−1; Figures 4b and S14). These observations indicated that the oxidation of Cyt c was favorable at higher [H+] (lower pH) conditions, in agreement with the mechanism of Cyt c oxidation and O2 reduction in a natural system. In summary, we showed that Cu2O NPs effectively receive electrons from Cyt c and exhibit CcO-like activity, which is dependent on the O2, pH, and Cu2O size. Cu2O NPs may act as catalytic centers where O2 is reduced to water. In a biological system, CcO uses a heme−copper center to perform its function by forming a complex system with Cyt c. Therefore, the CcO-like activity of Cu2O NPs in the presence of Cyt c closely mimics the native Cyt c−CcO enzyme system. Beyond the usual small organic molecules, this study suggests the potential application of Cu2O NPs in biochemistry and biotechnology in a complex with biomacromolecules. The enzyme-like activity of inorganic nanomaterials would open new vistas in enzyme construction, material chemistry, and related research fields.
nitrogen atmosphere and then exposed the system to air. Interestingly, it was found that the oxidation rate of ferrous Cyt c increased when exposed to air (Figure S9), which further proved the vital role of O2 in the oxidation of Cyt c. In addition, to examine the influence of the particle size on the CcO-like activity of Cu2O NPs, we prepared two other kinds of Cu2O NPs with sizes of ∼150 and ∼500 nm, respectively (details in the SI, Figures S10 and S11). The CcO-like activity studies showed that the oxidation of ferrous Cyt c with 150 and 500 nm Cu2O was slower than that with 50 nm Cu2O NPs (Figure 3a).
Figure 3. Cu2O size-dependent oxidation of Cyt c (phosphate buffer, pH 7.2). (a) Kinetic traces showing the decrease of reduced Cyt c (following the intensity at 550 nm). (b) Comparison of the rate constants of Cyt c oxidation catalyzed by different Cu2O NPs.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01393. General experimental procedures, additional experimental details, and additional data and references (PDF)
After 60 min of oxidation, about 77% and 56% of ferrous Cyt c were oxidized to its ferric state in the presence of ∼150 and ∼500 nm Cu2O NPs, with rate constants of 0.0263 and 0.0144 min−1, respectively, which were ∼68% and ∼38% of Cu2O NPs with a size of ∼50 nm (Figures 3b and S12). These observations indicated that the CcO-like activity of Cu2O NPs is sizedependent; that is, the smaller the particle size, the higher the catalytic activity. One possible interpretation of this observation is that smaller particles have a larger surface-to-volume ratio to interact with both Cyt c and O2 molecules, which facilitates the electron transfer from ferrous Cyt c to O2 molecules and accelerates the oxidation of Cyt c and reduction of O2. CD spectra in the 190−250 nm region provide additional evidence for the interaction between Cu2O NPs and Cyt c in both oxidation states, resulting in slight conformational changes (Figure S13). In biosystems, CcO catalyzes the 4e−/4H+ reduction of O2 to H2O by receiving electrons from ferrous Cyt c [4Cyt c(Fe2+) + O2 + 4H+→ 4Cyt c(Fe3+) + 2H2O] in the terminal step of the electron-transport chain during respiration.13 Therefore, in the artificial Cyt c−Cu2O NPs system, the solution pH may have an influence on the CcO-like activity. To examine this possibility, we performed oxidation of ferrous Cyt c by varying one pH unit from 7 with minimal conformational changes. As shown in Figure 4a,
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] or
[email protected] (Z.W.). *E-mail:
[email protected] (Y.-W.L.). ORCID
Zhonghua Wang: 0000-0002-3527-5405 Xiaohui Jiang: 0000-0002-9820-6060 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the Open Project of Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province (Grant CSPC2016-3-2), the Innovation Team Project of the Education Department of Sichuan Province (Grant 15TD0018), and the National Science Foundation of China (Grant 31370812).
■
REFERENCES
(1) Dong, Y. L.; Zhang, H. G.; Rahman, Z. U.; Su, L.; Chen, X. J.; Hu, J.; Chen, X. G. Graphene oxide-Fe3O4 magnetic nanocomposites with peroxidase-like activity for colorimetric detection of glucose. Nanoscale 2012, 4, 3969−3976. (2) Iyer, P. V.; Ananthanarayan, L. Enzyme stability and stabilization− aqueous and non-aqueous environment. Process Biochem. 2008, 43, 1019−1032. (3) Klotz, I. M.; Royer, G. P.; Scarpa, I. S. Synthetic derivatives of polyethyleneimine with enzyme-like catalytic activity (synzymes). Proc. Natl. Acad. Sci. U. S. A. 1971, 68, 263−264. (4) Lu, Y.; Yeung, N.; Sieracki, N.; Marshall, N. M. Design of functional metalloproteins. Nature 2009, 460, 855−862. (5) Wang, Z. H.; Ando, Y.; Nugraheni, A. D.; Ren, C. G.; Nagao, S.; Hirota, S. Self-oxidation of cytochrome c at methionine80 with
Figure 4. pH-dependent oxidation of Cyt c catalyzed by 50 nm Cu2O NPs. (a) Kinetic traces showing the decrease of reduced Cyt c (following the intensity at 550 nm). (b) Comparison of the rate constants of Cyt c oxidation at different pHs. 9402
DOI: 10.1021/acs.inorgchem.7b01393 Inorg. Chem. 2017, 56, 9400−9403
Communication
Inorganic Chemistry molecular oxygen induced by cleavage of the Met-heme iron bond. Mol. BioSyst. 2014, 10, 3130−3137. (6) Wei, H.; Wang, E. K. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 2013, 42, 6060−6093. (7) Ozaki, S.; Matsui, T.; Roach, M. P.; Watanabe, Y. Rational molecular design of a catalytic site: engineering of catalytic functions to the myoglobin active site framework. Coord. Chem. Rev. 2000, 198, 39− 59. (8) Wang, Z. H.; Lin, Y. W.; Rosell, F. I.; Ni, F. Y.; Lu, H. J.; Yang, P. Y.; Tan, X. S.; Li, X. Y.; Huang, Z. X.; Mauk, A. G. Converting cytochrome c into a peroxidase-like metalloenzyme by molecular design. ChemBioChem 2007, 8, 607−609. (9) Lin, Y.-W. Rational design of metalloenzymes: From single to multiple active sites. Coord. Chem. Rev. 2017, 336, 1−27. (10) Wang, X. Y.; Hu, Y. H.; Wei, H. Nanozymes in bionanotechnology: from sensing to therapeutics and beyond. Inorg. Chem. Front. 2016, 3, 41−60. (11) Cheng, H. J.; Zhang, L.; He, J.; Guo, W. J.; Zhou, Z. Y.; Zhang, X. J.; Nie, S. M.; Wei, H. Integrated nanozymes with nanoscale proximity for in vivo neurochemical monitoring in living brains. Anal. Chem. 2016, 88, 5489−5497. (12) Hu, Y.; Cheng, H.; Zhao, X.; Wu, J.; Muhammad, F.; Lin, S.; He, J.; Zhou, L.; Zhang, C.; Deng, Y.; Wang, P.; Zhou, Z.; Nie, S.; Wei, H. Surface-enhanced raman scattering active gold nanoparticles with enzyme-mimicking activities for measuring glucose and lactate in living tissues. ACS Nano 2017, 11, 5558−5566. (13) Ferguson-Miller, S.; Babcock, G. T. Heme/copper terminal oxidases. Chem. Rev. 1996, 96, 2889−2907. (14) Khalavka, Y.; Becker, J.; Sonnichsen, C. Synthesis of rod-shaped gold nanorattles with improved plasmon sensitivity and catalytic activity. J. Am. Chem. Soc. 2009, 131, 1871−1875. (15) Bell, A. T. The impact of nanoscience on heterogeneous catalysis. Science 2003, 299, 1688−1691. (16) Chen, J. P.; Patil, S.; Seal, S.; McGinnis, J. F. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 2006, 1, 142−150. (17) Xie, J. X.; Zhang, X. D.; Wang, H.; Zheng, H. Z.; Huang, Y. M.; Xie, J. X. Analytical and environmental applications of nanoparticles as enzyme mimetics. TrAC, Trends Anal. Chem. 2012, 39, 114−129. (18) Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577−583. (19) Qin, W. J.; Su, L.; Yang, C.; Ma, Y. H.; Zhang, H. J.; Chen, X. G. Colorimetric detection of sulfite in foods by a TMB-O2-Co3O4 nanoparticles detection system. J. Agric. Food Chem. 2014, 62, 5827− 5834. (20) Cheng, H.; Lin, S.; Muhammad, F.; Lin, Y.-W.; Wei, H. Rationally modulate the oxidase-like activity of nanoceria for self-regulated bioassays. ACS Sensors 2016, 1, 1336−1343. (21) Chen, M.; Shu, J.; Wang, Z.; Ren, C. Porous surface MnO2 microspheres as oxidase mimetics for colorimetric detection of sulfite. J. Porous Mater. 2017, 24, 973−977. (22) Wang, Z. H.; Chen, M.; Shu, J. X.; Li, Y. One-step solvothermal synthesis of Fe3O4@Cu@Cu2O nanocomposite as magnetically recyclable mimetic peroxidase. J. Alloys Compd. 2016, 682, 432−440. (23) Hayat, A.; Cunningham, J.; Bulbul, G.; Andreescu, S. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays. Anal. Chim. Acta 2015, 885, 140−147. (24) Dutta, A. K.; Das, S.; Samanta, S.; Samanta, P. K.; Adhikary, B.; Biswas, P. CuS nanoparticles as a mimic peroxidase for colorimetric estimation of human blood glucose level. Talanta 2013, 107, 361−367. (25) An, Q.; Sun, C. Y.; Li, D.; Xu, K.; Guo, J.; Wang, C. C. Peroxidaselike activity of Fe3O4@carbon nanoparticles enhances ascorbic acidinduced oxidative stress and selective damage to PC-3 prostate cancer cells. ACS Appl. Mater. Interfaces 2013, 5, 13248−13257.
(26) Chen, J. Z.; Liu, Y. J.; Zhu, G. X.; Yuan, A. H. Ag@Fe3O4 nanowire: fabrication, characterization and peroxidase-like activity. Cryst. Res. Technol. 2014, 49, 309−314. (27) Gou, L.; Murphy, C. J. Controlling the size of Cu2O nanocubes from 200 to 25 nm. J. Mater. Chem. 2004, 14, 735−738. (28) Shu, J. X.; Wang, Z. H.; Huang, Y. J.; Huang, N.; Ren, C. G.; Zhang, W. Adsorption removal of Congo red from aqueous solution by polyhedral Cu2O nanoparticles: Kinetics, isotherms, thermodynamics and mechanism analysis. J. Alloys Compd. 2015, 633, 338−346. (29) Zhang, Y. G.; Ma, L. L.; Li, J. L.; Yu, Y. In situ Fenton reagent generated from TiO2/Cu2O composite film: A new way to utilize TiO2 under visible light irradiation. Environ. Sci. Technol. 2007, 41, 6264− 6269. (30) Sun, W.; Sun, W. D.; Zhuo, Y. J.; Chu, Y. Facile synthesis of Cu2O nanocube/polycarbazole composites and their high visible-light photocatalytic properties. J. Solid State Chem. 2011, 184, 1638−1643. (31) Wang, W. Z.; Wang, G. H.; Wang, X. S.; Zhan, Y. J.; Liu, Y. K.; Zheng, C. L. Synthesis and characterization of Cu2O nanowires by a novel reduction route. Adv. Mater. 2002, 14, 67−69. (32) Huang, H.; Huang, N.; Wang, Z.; Xia, G.; Chen, M.; He, L.; Tong, Z.; Ren, C. Room-temperature synthesis of carnation-like ZnO@AgI hierarchical nanostructures assembled by AgI nanoparticles-decorated ZnO nanosheets with enhanced visible light photocatalytic activity. J. Colloid Interface Sci. 2017, 502, 77−88. (33) Francisco, M. S. P.; Mastelaro, V. R.; Nascente, P. A. P.; Florentino, A. O. Activity and characterization by XPS, HR-TEM, Raman spectroscopy, and BET surface area of CuO/CeO2-TiO2 catalysts. J. Phys. Chem. B 2001, 105, 10515−10522. (34) Ying, T.; Zhong, F.; Wang, Z. H.; Xie, J.; Tan, X.; Huang, Z. X. Generation of novel functional metalloproteins via hybrids of cytochrome c and peroxidase. Protein Eng., Des. Sel. 2013, 26, 401−407.
9403
DOI: 10.1021/acs.inorgchem.7b01393 Inorg. Chem. 2017, 56, 9400−9403
