Fine���tuning the reduction potential of Cupredoxin proteins by altering secondary coordination sphere interactions

Metalloenzyme;protein engineering;electron transfer;redox potential

Marshall, Nicholas M.

关键词: Metalloenzyme;    protein engineering;    electron transfer;    redox potential;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/26396/Marshall_Nicholas.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Electron transfer (ET) reactions are at the heart of most biological and chemicalprocesses. This seemingly simple passing of electrons is critical for all life, as it allows forphotosynthesis, respiration, synthesis of molecules, etc. This myriad of biological processesrequires a similarly diverse set of ET catalysts to carry and transfer the electrons in a selectivefashion. The reduction potentials (Em) of these protein based catalysts span the full range ofbiologically relevant Em values from about ‐500 mV to 1,000 mV. Despite the diversity inreactivity and Em seen in natural ET catalysts, these proteins utilize only a limited number ofredox active cofactors. Of metal based redox cofactors, the entirety of the required Em range iscovered 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 maybe effectively identical, yet Em values within a family of ET protein may vary 500 mV or more. Astriking example of this is the type 1 (T1) Cupredoxin family of proteins. In this family ofproteins, Em values vary across 500‐600 mV, but the redox active copper is ligated in a similardistorted tetrahedral fashion, with identical ligands. The spectroscopic features of theseproteins are also very similar across the entire family. More importantly, the unchanged redoxactive site is able to maintain a low reorganization energy associated with reduction oroxidation, which manifests as highly efficient ET. How the Em values of these proteins can vary sogreatly and in very small, ~50 mV increments, with seemingly little to no change to the redoxsite itself is one of the most critical and previously unanswered questions in the field ofbiological ET.This thesis details efforts to answer how the Em of a single ET site can vary over an 500mV 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, ~50iiimV increments across a 500‐600 mV range simply by modifying hydrogen bonding andhydrophobic 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 theprotein. It is also shown that the effects on the Em of certain individual mutations is additive andthat these individual mutations can be combined to increase the range of attainable redoxpotentials. Most importantly, these mutations do not significantly perturb the ET efficiency or ETrate of the protein.Hydrogen bonding and hydrophobic interactions are, of course, not the only featureswithin a protein that could alter the Em. The contribution of a negative dipole from a carbonyloxygen 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 proteinsequence it cannot be directly changed by traditional mutagenesis or even semi‐syntheticmethods. As such, hydrogen bonding, steric repulsion and loop torsion were used in attempts toalter the backbone carbonyl and investigate its electronic contribution to the copper site. It wasseen that this region of the protein, and presumably the carbonyl oxygen itself, does have astrong effect on the Em of Az, with alteration of hydrogen bonding producing the largest changesin Em.The individual mutations giving the largest increases to the Em of Az discoveredthroughout this study were then combined into a single Az variant, resulting in a copper sitewith an Em approaching 1,000 mV. This value is well above the highest known Em value for aprotein similar to Az, but not unprecedented in nature. Unfortunately the mutations made togenerate the 1,000 mV variant did alter the spectroscopy and, presumably, the ET efficiency ofthe copper site. Given the strongly oxidizing Em of this protein and others in the range leading tomaking the 1,000 mV variant, the viability of using such an Az variant as a redox reagent forivoxidizing other proteins was investigated. Preliminary results show that heme proteins likecytochrome p450 camphor can be oxidized with Az to generate a catalytic species capable ofoxidizing organic substrates.Secondly, this thesis also shows how the lessons learned from redox tuning of Az can beapplied to other metal sites. The Em of the dinuclear CuA site in the engineered protein CuA Azwas tuned in a similar fashion by altering the hydrogen bonding network around one of thecopper ligands. Because > 85 % of the protein is the same between Az with a T1 copper site andCuA Az with a dinuclear copper site, many of the alternate effects of the mutations could bediscounted. It was shown that the two types of copper sites can be redox tuned in effectivelythe same fashion, but that the electronic properties of the CuA site are more spread out whichlessens the change in Em observed upon mutation.Finally, initial investigations and characterization of newly discovered cupredoxinproteins from the organism Nitrosopumilus maritimus will be discussed. This organism isinteresting as it and similar organisms are believed to account for much of the global conversionof ammonia in the world’s oceans to nitrite. Interestingly, Nitrosopumilus maritimus is alsomissing 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 thecopper proteins from this organism will undoubtedly shed light on the mechanisms of ammoniaoxidation in this organism, but will also shed light onto the evolutionary question of why naturechose iron as its metal of choice throughout most of life, rather than copper. One of the copperproteins 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 alreadyresulted in some interesting proposals as to the function of this novel copper protein.vIn summary, this thesis details in depth studies of the secondary coordination sphereinteractions 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 nearly1,000 mV range. The strongly oxidizing variants of Az developed here may also be useful asoxidants for other proteins. Interactions used to alter the Em in Az were also applied to a CuA siteand results show that the effects of individual mutations are the same across different metalsites, although the magnitude of the change is difficult to predict. Finally, the properties andfunctions of newly discovered copper proteins, which may be involved in the global nitrogencycle, are being investigated.