Characterizing a structural and functional model of nitric oxide reductase performing NO and O2 reduction in engineered myoglobin

Reed, Julian H.

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https://hdl.handle.net/2142/99473 Copy

Description

Title

Characterizing a structural and functional model of nitric oxide reductase performing NO and O2 reduction in engineered myoglobin

Author(s)

Reed, Julian H.

Issue Date

2017-10-30

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Gennis, Robert
• Procko, Erik
• Sligar, Stephen

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Nitric oxide reductase (NOR)
• FeBMb
• Nitrogenase
• Cytochrome c oxidase
• Heme-copper oxidase (HCO)

Abstract

Nitric oxide reductase (NOR) is a membrane-bound metalloenzyme in the denitrification pathway of many bacteria. NORs are structurally homologous to subunit I of HCOs (heme-copper oxidases); however, the copper site (CuB) in HCOs is replaced with a non-heme iron site (FeB) in NOR, and each metal contains a different primary and secondary coordination sphere.1 NOR and HCO also have cross-reactivity, though they are more reactive toward their native substrate. NOR is capable of O2 reduction and some members of the HCO superfamily are capable of NO reduction.2–4 Therefore, an important question is whether the identity of the metal helps tune activity in homologous protein metal binding sites, namely the CuB site in HCO and the FeB site in NOR. A CuB site was engineered into the scaffold protein myoglobin (CuBMb).5 However, FeII binding was not observed in CuBMb. To mimic the conserved glutamate(s) in NOR, and aim to also bind FeII, a glutamate was introduced near the heme of CuBMb, called FeBMb. Crystallographic and/or spectroscopic studies showed that FeBMb could bind Fe or Cu in its engineered FeB site, and was observed to be both a structural and functional model of native NORs through multiple generations of mutants6,7 Still remaining to be understood are detailed studies about the particular roles that specific designed features play in conferring and fine-tuning the different reactivities of FeBMb. This thesis describes work utilizing FeBMb to better describe the role of structural features, such as nonheme metal ion, in conferring biologically significant activities such as NO and O2 reduction. A detailed understanding of the role that nonheme Fe plays in NOR reactivity in FeBMb was previously unexplored, but replacement of Fe-PPIX with Zn-PPIX allowed for detailed spectroscopic characterization of the nonheme Fe-nitrosyl species.8 Enrichment of each site with 57Fe allowed for advanced NRVS measurements of all vibrational modes of the 57Fe sites, as well as any 57Fe-nitrosyl species, important for understanding mechanistic intermediates in the NOR reaction.9 Unique studies of the reactivity of FeBMb performing both NO and O2 reduction while utilizing nonheme Fe, Mn, Co, and Cu were also carried out. Finally, work aimed at engineering more complex metal binding sites, such as those for the FeMo cofactor of nitrogenase, utilizing the Rosetta software suite are described in detail. Insights gained from the study of FeBMb and its relevance to NOR and HCO include evidence to support the so called trans mechanism of NO reduction, whereby 2 molecules of NO bind at the FeBMb active site, one to each Fe, before N2O formation. Additionally, NRVS measurements demonstrated site-specific 57Fe-labelling of both the heme and nonheme sites of FeBMb, and identified nitrosyl species for each. Replacement of nonheme Fe with other metals, such as Mn and Co, identified a nonheme metal redox-inactive mechanism of O2 reduction in FeBMb. These results demonstrate that the use of small, easy to characterize, experimentally tractable mimics of larger, more complex native metalloenzymes provides a wealth of knowledge to help guide future research and uncover interesting discoveries that would otherwise be lost.

Graduation Semester

2017-12

Type of Resource

text

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http://hdl.handle.net/2142/99473

Copyright and License Information

Copyright 2017 Julian Reed

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