{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/102876"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/102876","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Engineering heme-copper and multi-copper oxidases for efficient oxygen reduction catalysis","abstract":"Fuel cell draws increasing attention because of its high theoretical efficiency (over 90%). However, the actual efficiency is limited by the large overpotential (500-600 mV) of the oxygen reduction reaction (ORR), which demands a potent ORR catalyst to decrease the overpotential. Towards this goal, my thesis focuses on mimicking the heme-copper oxidase and engineering a multi-copper oxidase for efficient oxygen reduction with high turnover frequency and low overpotential Heme-copper oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s−1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s−1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells. The other system we studied is a multi-copper oxidase called small laccase (SLAC), which consists of a catalytic trinuclear copper center (TNC) and a Type 1 (T1) Cu center for electron transfer. However, the ORR overpotential of SLAC is relatively high (400 mV) because of the lower reduction potential (Eo') of the T1 Cu (370 mV) compared to fungal laccases (780 mV). Built upon our previous success in tuning reduction potentials of T1 Cu protein azurin, we have made significant progress in tuning the T1 Cu center in SLAC to decrease its over-potentials. We achieved the goal by structural overlays of the T1 Cu domain of SLAC and the high potential azurin reported by Lu group to search for mutations that can potentially increase the Eo' of T1 Cu through non-covalent secondary sphere interactions such as dipole interactions and hydrophobicity and validated the hypothesis that the Eo' of T1 Cu plays a crucial role in defining the onset potential of ORR. The results provide a viable approach to decrease the ORR overpotential for biofuel cell application. Additionally, we studied the role of the axial methionine in tuning the onset potential of small laccase. We observed a positive correlation between the onset potential of oxygen reduction and the A// hyperfine of the T1 copper. The hyperfine coupling originates from the coupling between Cu nuclei and electron spin and a greater hyperfine coupling indicates a larger spin density localized on the Cu center, i.e., the Cu is more electron deficient resulting in a higher reduction potential. In summary, we achieved the oxidase activity equivalent to that of native heme-copper oxidase in a designed metalloenzyme and attained a 180 mV overpotential with a rationally design triple mutant of small laccase. This work demonstrated the redox tuning of a multi-copper oxidase by non-covalent interactions and provided a viable approach to decrease the ORR overpotential by tuning the Eo' of T1 Cu for biofuel cell application.","abstract_html":"Fuel cell draws increasing attention because of its high theoretical efficiency (over 90%). However, the actual efficiency is limited by the large overpotential (500-600 mV) of the oxygen reduction reaction (ORR), which demands a potent ORR catalyst to decrease the overpotential. Towards this goal, my thesis focuses on mimicking the heme-copper oxidase and engineering a multi-copper oxidase for efficient oxygen reduction with high turnover frequency and low overpotential Heme-copper oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s−1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s−1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells. The other system we studied is a multi-copper oxidase called small laccase (SLAC), which consists of a catalytic trinuclear copper center (TNC) and a Type 1 (T1) Cu center for electron transfer. However, the ORR overpotential of SLAC is relatively high (400 mV) because of the lower reduction potential (Eo') of the T1 Cu (370 mV) compared to fungal laccases (780 mV). Built upon our previous success in tuning reduction potentials of T1 Cu protein azurin, we have made significant progress in tuning the T1 Cu center in SLAC to decrease its over-potentials. We achieved the goal by structural overlays of the T1 Cu domain of SLAC and the high potential azurin reported by Lu group to search for mutations that can potentially increase the Eo' of T1 Cu through non-covalent secondary sphere interactions such as dipole interactions and hydrophobicity and validated the hypothesis that the Eo' of T1 Cu plays a crucial role in defining the onset potential of ORR. The results provide a viable approach to decrease the ORR overpotential for biofuel cell application. Additionally, we studied the role of the axial methionine in tuning the onset potential of small laccase. We observed a positive correlation between the onset potential of oxygen reduction and the A// hyperfine of the T1 copper. The hyperfine coupling originates from the coupling between Cu nuclei and electron spin and a greater hyperfine coupling indicates a larger spin density localized on the Cu center, i.e., the Cu is more electron deficient resulting in a higher reduction potential. In summary, we achieved the oxidase activity equivalent to that of native heme-copper oxidase in a designed metalloenzyme and attained a 180 mV overpotential with a rationally design triple mutant of small laccase. This work demonstrated the redox tuning of a multi-copper oxidase by non-covalent interactions and provided a viable approach to decrease the ORR overpotential by tuning the Eo' of T1 Cu for biofuel cell application.","abstract_has_math":false,"creators":["Cui, Chang"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Lu, Yi","Suslick, Kenneth S.","Yang, Hong","Vura-Weis, Josh"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-02-08T18:39:36Z","date_published":"2019-02-08T18:39:36Z","updated_at":"2026-07-22T22:24:42Z","subjects":["oxygen reduction reaction, multi-copper oxidase"],"languages":["en"],"rights":["Copyright 2018 Chang Cui"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/102876","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Lu, Yi","Suslick, Kenneth S.","Yang, Hong","Vura-Weis, Josh"]},{"key":"dc:creator","label":"Author","values":["Cui, Chang"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-02-08T18:39:36Z","2021-02-09T10:15:21Z","2018-07-19","2018-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["oxygen reduction reaction, multi-copper oxidase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2018 Chang Cui"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/102876"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Fuel cell draws increasing attention because of its high theoretical efficiency (over 90%). However, the actual efficiency is limited by the large overpotential (500-600 mV) of the oxygen reduction reaction (ORR), which demands a potent ORR catalyst to decrease the overpotential. Towards this goal, my thesis focuses on mimicking the heme-copper oxidase and engineering a multi-copper oxidase for efficient oxygen reduction with high turnover frequency and low overpotential Heme-copper oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s−1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s−1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells. The other system we studied is a multi-copper oxidase called small laccase (SLAC), which consists of a catalytic trinuclear copper center (TNC) and a Type 1 (T1) Cu center for electron transfer. However, the ORR overpotential of SLAC is relatively high (400 mV) because of the lower reduction potential (Eo') of the T1 Cu (370 mV) compared to fungal laccases (780 mV). Built upon our previous success in tuning reduction potentials of T1 Cu protein azurin, we have made significant progress in tuning the T1 Cu center in SLAC to decrease its over-potentials. We achieved the goal by structural overlays of the T1 Cu domain of SLAC and the high potential azurin reported by Lu group to search for mutations that can potentially increase the Eo' of T1 Cu through non-covalent secondary sphere interactions such as dipole interactions and hydrophobicity and validated the hypothesis that the Eo' of T1 Cu plays a crucial role in defining the onset potential of ORR. The results provide a viable approach to decrease the ORR overpotential for biofuel cell application. Additionally, we studied the role of the axial methionine in tuning the onset potential of small laccase. We observed a positive correlation between the onset potential of oxygen reduction and the A// hyperfine of the T1 copper. The hyperfine coupling originates from the coupling between Cu nuclei and electron spin and a greater hyperfine coupling indicates a larger spin density localized on the Cu center, i.e., the Cu is more electron deficient resulting in a higher reduction potential. In summary, we achieved the oxidase activity equivalent to that of native heme-copper oxidase in a designed metalloenzyme and attained a 180 mV overpotential with a rationally design triple mutant of small laccase. This work demonstrated the redox tuning of a multi-copper oxidase by non-covalent interactions and provided a viable approach to decrease the ORR overpotential by tuning the Eo' of T1 Cu for biofuel cell application.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-12-01","The student, Chang Cui, accepted the attached license on 2018-07-18 at 10:09.","The student, Chang Cui, submitted this Dissertation for approval on 2018-07-18 at 10:13.","This Dissertation was approved for publication on 2018-07-19 at 16:26.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12917 on 2019-02-08 at 11:37:26","Made available in DSpace on 2019-02-08T18:39:36Z (GMT). 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However, the actual efficiency is limited by the large overpotential (500-600 mV) of the oxygen reduction reaction (ORR), which demands a potent ORR catalyst to decrease the overpotential. Towards this goal, my thesis focuses on mimicking the heme-copper oxidase and engineering a multi-copper oxidase for efficient oxygen reduction with high turnover frequency and low overpotential Heme-copper oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s−1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s−1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells. The other system we studied is a multi-copper oxidase called small laccase (SLAC), which consists of a catalytic trinuclear copper center (TNC) and a Type 1 (T1) Cu center for electron transfer. However, the ORR overpotential of SLAC is relatively high (400 mV) because of the lower reduction potential (Eo') of the T1 Cu (370 mV) compared to fungal laccases (780 mV). Built upon our previous success in tuning reduction potentials of T1 Cu protein azurin, we have made significant progress in tuning the T1 Cu center in SLAC to decrease its over-potentials. We achieved the goal by structural overlays of the T1 Cu domain of SLAC and the high potential azurin reported by Lu group to search for mutations that can potentially increase the Eo' of T1 Cu through non-covalent secondary sphere interactions such as dipole interactions and hydrophobicity and validated the hypothesis that the Eo' of T1 Cu plays a crucial role in defining the onset potential of ORR. The results provide a viable approach to decrease the ORR overpotential for biofuel cell application. Additionally, we studied the role of the axial methionine in tuning the onset potential of small laccase. We observed a positive correlation between the onset potential of oxygen reduction and the A// hyperfine of the T1 copper. The hyperfine coupling originates from the coupling between Cu nuclei and electron spin and a greater hyperfine coupling indicates a larger spin density localized on the Cu center, i.e., the Cu is more electron deficient resulting in a higher reduction potential. In summary, we achieved the oxidase activity equivalent to that of native heme-copper oxidase in a designed metalloenzyme and attained a 180 mV overpotential with a rationally design triple mutant of small laccase. This work demonstrated the redox tuning of a multi-copper oxidase by non-covalent interactions and provided a viable approach to decrease the ORR overpotential by tuning the Eo' of T1 Cu for biofuel cell application.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-12-01","The student, Chang Cui, accepted the attached license on 2018-07-18 at 10:09.","The student, Chang Cui, submitted this Dissertation for approval on 2018-07-18 at 10:13.","This Dissertation was approved for publication on 2018-07-19 at 16:26.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12917 on 2019-02-08 at 11:37:26","Made available in DSpace on 2019-02-08T18:39:36Z (GMT). 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