SPECTROSCOPIC AND THEORETICAL STUDIES OF T1 CU, CUA AND CYTOCHROME C: GEOMETRIC AND ELECTRONIC STRUCTURE CONTRIBUTIONS TO ELECTRON TRANSFER A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CHEMISTRY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Xiangjin Xie January 2010 © 2010 by Xie Xiangjin. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/cq701yv2053 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Edward Solomon, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. T Stack I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Robert Waymouth Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT Metal sites that are known to be involved in biological electron transfer (ET) include Type 1 Copper (T1 Cu), CuA, cytochromes, and the 1-, 2-, 3-, and 4-iron sulfur centers (rubredoxin, ferredoxins, and high potential iron-sulfur proteins (HiPIPs)). These ET sites generally exhibit unusual spectroscopic features reflecting novel geometric and electronic structures that contribute to function. My focuses are on T1 Cu, CuA, cytochrome c proteins utilizing a wide-range of spectroscopies combined with density functional calculations to understand active site electronic structures, the origin of their geometric structures, and possible contributions to function. Five major achievements are: 1) defined the temperature dependent absorption feature of T1 Cu site in nitrite reductase (NIR) and provided insight into the entatic/rack nature of the blue Cu site in plastocyanin; 2) addressed the interesting absorption features of the T1 Cu site in P. pantotrophus pseudoazurin and demonstrated the spectral probes of the weak axial ligation in metalloprotein; 3) resolved a two-state issue in the mixed-valence binuclear CuA centers in cytochrome c oxidases (CcO) and nitrous oxide reductases (N2O) by a combination of density functional calculations and spectroscopy analyses, and evaluated proteins role in CuA sites and their contributions to ET function; 4) determined that the Cu-Cu interaction in CuA keeps the site delocalized even upon loss of a Histidine (NHis) ligand due to protonation, and defined the contribution of σ delocalization to efficient ET; 5) investigated the nature of the Fe-SMet bond in ferricytochrome c. (1) Thermodynamic Equilibrium between Blue and Green Copper Sites and the Role of the Protein in Controlling Function Spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (ΔH ≈ 4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TΔS ≈4.5 kcal/mol, at room iv temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature- dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by > 200 mV. (2) Variable Temperature Spectroscopic Study on Pseudoazurin: Effects of Protein Constraints on the Blue Cu Site. The T1 copper site of Paracoccus pantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are σ and π SCys to Cu2+ charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase, which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 Å. However, plastocyanin exhibits only π CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thioether in pseudoazurin is also constrained, but at a shorter 2+ Cu–SMet bond length. This leads to an increase in the Cu –SCys bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state 2+ wavefunction has both σ and π character in the Cu –SCys bond. (3) The Two State Issue in the Mixed-Valence Binuclear CuA Center in Cytochrome c Oxidase and N2O Reductase For the CuA site in the protein, the ground and lowest energy excited-states are σu* and πu, respectively, denoting the types of Cu-Cu interactions. EPR data on CuA proteins show a low g|| value of 2.19 deriving from spin-orbital coupling between σu* and πu, -1 which requires an energy gap between σu* and πu of 3000−4500 cm . On the other hand, v from paramagnetic NMR studies, it has been observed that the first excited-state is thermally accessible and the energy gap between the ground state and the thermally accessible state is 350 cm-1. This study addressed this apparent discrepancy and evaluated the roles of the two electronic states, σu* and πu, in electron transfer (ET) of CuA. The potential energy surface calculations show that both NMR and EPR results are consistent within the electronic/geometric structure of CuA. The anti-Curie behavior observed in paramagnetic NMR studies of CuA results from the thermal equilibrium between the σu* and πu states, which are at very close energies in their respective equilibrium geometries. Alternatively, the EPR g-value analysis involves the σu* ground state in the geometry with a short dCu-Cu where the πu state is a Frank−Condon excited- state with the energy of 3200 cm-1. The protein environment plays a role in maintaining CuA in the σu* state as a lowest-energy state with the lowest reorganization energy and high-covalent coupling to the Cys and His ligands for efficient intra- and intermolecular ET with a low-driving force. (4) Perturbations to the Geometric and Electronic Structure of the CuA Site: Factors that Influence Delocalization and their Contributions to Electron Transfer Using a combination of electronic spectroscopies and DFT calculations, the effect of pH perturbation on the geometric and electronic structure of the CuA site has been defined. Descriptions are developed for high pH (pH = 7) and low pH (pH = 4) forms of CuA azurin and its H120A mutant which address the discrepancies concerning the extent of delocalization indicated by multifrequency EPR and ENDOR data. Our resonance Raman and MCD spectra demonstrate that the low pH and H120A mutant forms are essentially identical and are the perturbed forms of the completely delocalized high pH CuA site. However, in going from high pH to low pH, a seven-line hyperfine coupling pattern associated with complete delocalization of the electron (S = 1/2) over two Cu coppers (ICu = 3/2) changes into a four-line pattern reflecting apparent localization. DFT calculations show that the unpaired electron is delocalized in the low pH form and reveal that its four-line hyperfine pattern results from the large EPR spectral effects of 1% 4s orbital contribution of one Cu to the ground-state spin wave function upon protonative loss of its His ligand. The contribution of the Cu−Cu interaction to electron vi delocalization in this low symmetry protein site is evaluated, and the possible functional significance of the pH-dependent transition in regulating proton-coupled electron transfer in cytochrome c oxidase is discussed. (5) The Fe-Smet Bond in Ferricytochrome c DFT calculations calibrated with experiment data were used to define the nature of the Fe-SMet bond in ferricytochrome c. This is inspired by the studies of NiR. vii ACKNOWLEDGMENTS To my research advisor Prof. Edward I. Solomon, whose deep insight in science, great passion in research, and thoughtful care in heart have been driving and elevating my work into a doctoral thesis. I am deeply impressed by his knowledge on numerous areas of research, and benefit tremendously from him. I feel highly grateful for his constant support and mentoring over the years. I am thankful to my dissertation committee members for their help and support taking time out of their busy schedules to read my thesis and be on my defense committee. I thank my collaborators Prof. Keith Hodgson, Prof. Britt Hedman, Dr. Ritimukta Sarangi and Munzarin Fatema Qayyum on CuA projects. Thanks are also due to Prof.