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Title:Fine���tuning the reduction potential of Cupredoxin proteins by altering secondary coordination sphere interactions
Author(s):Marshall, Nicholas M.
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Gennis, Robert B.; Luthey-Schulten, Zaida A.; Rauchfuss, Thomas B.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
protein engineering
electron transfer
redox potential
Abstract:Electron transfer (ET) reactions are at the heart of most biological and chemical processes. This seemingly simple passing of electrons is critical for all life, as it allows for photosynthesis, respiration, synthesis of molecules, etc. This myriad of biological processes requires a similarly diverse set of ET catalysts to carry and transfer the electrons in a selective fashion. The reduction potentials (Em) of these protein based catalysts span the full range of biologically relevant Em values from about ‐500 mV to 1,000 mV. Despite the diversity in reactivity and Em seen in natural ET catalysts, these proteins utilize only a limited number of redox active cofactors. Of metal based redox cofactors, the entirety of the required Em range is covered by Fe‐S clusters, heme, non‐heme iron, copper, manganese and molybdenum ions. Many of these cofactors are also so similar to one another that the spectroscopic features may be effectively identical, yet Em values within a family of ET protein may vary 500 mV or more. A striking example of this is the type 1 (T1) Cupredoxin family of proteins. In this family of proteins, Em values vary across 500‐600 mV, but the redox active copper is ligated in a similar distorted tetrahedral fashion, with identical ligands. The spectroscopic features of these proteins are also very similar across the entire family. More importantly, the unchanged redox active site is able to maintain a low reorganization energy associated with reduction or oxidation, which manifests as highly efficient ET. How the Em values of these proteins can vary so greatly and in very small, ~50 mV increments, with seemingly little to no change to the redox site itself is one of the most critical and previously unanswered questions in the field of biological ET. This thesis details efforts to answer how the Em of a single ET site can vary over an 500 mV or larger range, yet maintain a very similar redox active site with similarly high ET efficiency. Firstly, it will be shown that the Em of the cupredoxin azurin (Az) can be fine‐tuned in small, ~50 iii mV increments across a 500‐600 mV range simply by modifying hydrogen bonding and hydrophobic interactions in the secondary coordination sphere of the copper site. Surprisingly, this large range of achievable Em values was attained with only two to three mutations to the protein. It is also shown that the effects on the Em of certain individual mutations is additive and that these individual mutations can be combined to increase the range of attainable redox potentials. Most importantly, these mutations do not significantly perturb the ET efficiency or ET rate of the protein. Hydrogen bonding and hydrophobic interactions are, of course, not the only features within a protein that could alter the Em. The contribution of a negative dipole from a carbonyl oxygen on the backbone of the protein towards the tuning of the Em of Az was also investigated. Since this dipole comes from a backbone oxygen atom that is in the middle of the protein sequence it cannot be directly changed by traditional mutagenesis or even semi‐synthetic methods. As such, hydrogen bonding, steric repulsion and loop torsion were used in attempts to alter the backbone carbonyl and investigate its electronic contribution to the copper site. It was seen that this region of the protein, and presumably the carbonyl oxygen itself, does have a strong effect on the Em of Az, with alteration of hydrogen bonding producing the largest changes in Em. The individual mutations giving the largest increases to the Em of Az discovered throughout this study were then combined into a single Az variant, resulting in a copper site with an Em approaching 1,000 mV. This value is well above the highest known Em value for a protein similar to Az, but not unprecedented in nature. Unfortunately the mutations made to generate the 1,000 mV variant did alter the spectroscopy and, presumably, the ET efficiency of the copper site. Given the strongly oxidizing Em of this protein and others in the range leading to making the 1,000 mV variant, the viability of using such an Az variant as a redox reagent for iv oxidizing other proteins was investigated. Preliminary results show that heme proteins like cytochrome p450 camphor can be oxidized with Az to generate a catalytic species capable of oxidizing organic substrates. Secondly, this thesis also shows how the lessons learned from redox tuning of Az can be applied to other metal sites. The Em of the dinuclear CuA site in the engineered protein CuA Az was tuned in a similar fashion by altering the hydrogen bonding network around one of the copper ligands. Because > 85 % of the protein is the same between Az with a T1 copper site and CuA Az with a dinuclear copper site, many of the alternate effects of the mutations could be discounted. It was shown that the two types of copper sites can be redox tuned in effectively the same fashion, but that the electronic properties of the CuA site are more spread out which lessens the change in Em observed upon mutation. Finally, initial investigations and characterization of newly discovered cupredoxin proteins from the organism Nitrosopumilus maritimus will be discussed. This organism is interesting as it and similar organisms are believed to account for much of the global conversion of ammonia in the world’s oceans to nitrite. Interestingly, Nitrosopumilus maritimus is also missing the genes encoding one of the most ubiquitous classes of ET proteins in nature, cytochrome c proteins. These genes are replaced by putative cupredoxin proteins. Studying the copper proteins from this organism will undoubtedly shed light on the mechanisms of ammonia oxidation in this organism, but will also shed light onto the evolutionary question of why nature chose iron as its metal of choice throughout most of life, rather than copper. One of the copper proteins of interest, N. mar_1307, has been purified and re‐constituted with copper. Spectroscopic and structural investigations of this protein are on‐going, but have already resulted in some interesting proposals as to the function of this novel copper protein. v In summary, this thesis details in depth studies of the secondary coordination sphere interactions that fine‐tune the Em of the cupredoxin Az. These interactions, hydrogen bonding, hydrophobicity and ionic interactions, allow for predictable tuning of the Em of Az across a nearly 1,000 mV range. The strongly oxidizing variants of Az developed here may also be useful as oxidants for other proteins. Interactions used to alter the Em in Az were also applied to a CuA site and results show that the effects of individual mutations are the same across different metal sites, although the magnitude of the change is difficult to predict. Finally, the properties and functions of newly discovered copper proteins, which may be involved in the global nitrogen cycle, are being investigated.
Issue Date:2011-08-26
Rights Information:Copyright 2011 Nicholas Marshall
Date Available in IDEALS:2013-08-27
Date Deposited:2011-08

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