Spectroscopic and computational elucidation of active sites in biological electron transfer and heterogeneous catalysis [electronic resource]
Keywords: Chemistry.

This thesis involves two areas of research. The first section presents studies related to metalloproteins involved in biological electron transfer (ET), while the second section presents studies on reaction intermediates and their formation in the zeolite Cu-ZSM-5. The three classes of metalloproteins that carry out biological ET include: 1) the mononuclear copper (Cu) type one (T1) and binuclear CuA, 2) the iron-sulfur clusters, and 3) the cytochromes. The encapsulation of the metal ion within the protein matrix, coupled to the presence of highly covalent bonding environments, results in the complex geometric and electronic structures of these active sites, which give rise to unique spectral features. These represent direct probes of the active site ligand-metal bonding and further reflect the interaction of the metal with the first-sphere ligands and the surrounding protein environment and solvent (i.e., H2O). Site-directed mutagenesis allows for these to be directly perturbed. A general approach here is the use of the spectral features and their perturbations to calibrate electronic structure calculations (i.e., density functional theory (DFT)) to understand how the first- and second-sphere protein environment both tune the properties of the metal and thus function. Chapters 2 -- 7 of the thesis are focused on the T1 Cu and CuA active sites. In Chapter 2, studies of the T1 Cu site of Paracoccus pantotrophus pseudoazurin (PAz) have shed light on the nature of the 'entatic/rack state' in T1 Cu proteins. In particular, PAz exhibits significant absorption intensity in both the 450 and 600 nm regions. These are S(Cys)p(σ) and S(Cys)p(π) → Cu(II) charge transfer (CT) transitions. In addition, the spectral features of PAz are temperature independent and reflect a single species. This contrasts the strongly temperature dependent behavior of the T1 center in Rhodobacter sphaeroides nitrite reductase (NiR), which has a S(Met) axial ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in PAz indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin (Pc) where the S(Met) ligand is constrained at 2.8 Å. However, Pc exhibits only π CT. This spectral difference between PAz and Pc reflects a coupled distortion of the site where the axial S(Met) in PAz is also constrained, but at a shorter Cu--S(Met) bond length. This leads to an increase in the Cu(II)--S(Cys) bond length and a partial tetragonal distortion of the T1 site in PAz. Thus, its ground state wavefunction has both σ and π character in the Cu(II)--S(Cys) bond. In Chapter 3, The rR data for the S(Cys)σ and S(Cys)π excited states show very different intensity distribution patterns for the vibrations in the 300--500 cm−1 region. Time-dependent DFT (TDDFT) calculations have been used to understand this difference and was shown to reflect the differential enhancement of S(Cys) backbone modes with Cu--S(Cys)--Cβ out-of-plane (oop) and in-plane bend character in their respective potential energy distributions. The rR excited state distortions have been related to ground state reorganization energies (λs) and predict that, in addition to M--L stretches, the Cu--S(Cys)--Cβ oop bend contributes. DFT calculations predict a large distortion in this bending coordinate upon reduction of a T1 site; however, this distortion is not present in the X-ray crystal structures of reduced T1 sites. The lack of Cu--S(Cys)--Cβ oop distortion upon reduction corresponds to a constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites. In Chapter 4, the role of the second-sphere protein environment in tuning E0 is elucidated for T1 Cu sites. The reduction potentials of T1 Cu sites in proteins and enzymes with identical first coordination spheres around the redox active Cu can vary by ~400 mV. Spectroscopic and DFT studies of a series of second-sphere variants—F114P, N47S, and F114N in Az—which modulate hydrogen bonding to and protein-derived dipoles nearby the Cu--S(Cys) bond have allowed for the fractionation of the contributions to tuning E0 into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active site interactions, while affording insight into the second-sphere mechanisms available to the protein to tune the E0 of ET sites in biology. In Chapter 5, a Cu--sulfenate complex is formed at the T1 Cu site of M121G Az and has been spectroscopically characterized. This species results from the reaction of H2O2 with Cu(I)--M121G Az. The presence of a side-on Cu--sulfenate species is supported by rR spectroscopy, electrospray mass spectrometry using isotopically enriched H2O2, and DFT calculations correlated to experiment. These studies indicate that the second-sphere interactions identified in Chapter 4 are critical in stabilizing this highly reactive species. Thus, the ET protein Az has been engineered into an active Cu enzyme that forms a biologically relevant Cu--sulfenate intermediate; demonstrating the importance of second-sphere interactions in stabilizing this species constitutes an important contribution toward understanding metal--sulfenate species in biology. Clearly, the axial ligand in T1 Cu proteins exerts a high degree of control over function, where blue copper (BC) has a weak axial S(Met) and green copper has a strong axial S(Met) ligand. CuA active sites also have a conserved S(Met) axial ligand. The role of this ligand in ET is the focus of Chapter 6. The mutation of the axial Met to Leu in a CuA site engineered into azurin (CuA Az) was found to have a limited effect on E0 relative to this mutation in BC. Spectroscopic and DFT studies on CuA Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. DFT calculations correlated to the spectral data show that the smaller ΔE0 is attributed to H2O coordination to the Cu center in the M123L mutant in CuA but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in CuA and BC (CuA ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E0 similarly in both. Electron delocalization over two Cu centers in CuA was found to minimize the Jahn--Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu--S(Met) bond in oxidized CuA is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E0 while maintaining a low reorganization energy required for rapid ET under physiological conditions. Two classes of enzymes contain T1 Cu sites that are covalently linked to catalytic metal active sites through a protein derived Cys-His bridge. These classes include the NiRs and the multicopper oxidases (MCOs). The NiRs are involved in bacterial denitrification and contain T1 Cu and type 2 (T2) Cu active sites. The T1 Cu center shuttles electrons via the Cys-His pathway to the T2 Cu active site where the one electron reduction of NO2- to NO and H2O occurs. The multicopper oxidases (MCOs) contain a T1 Cu active site that shuttles electrons to a trinuclear Cu cluster where O2 is reduced to H2O. This process is coupled to the oxidation of a wide variety of substrates as a source of the reducing equivalents. It is interesting to consider that native NiRs function with both blue (Sp(π)) and green (Sp(σ)) T1 Cu sites while the MCOs only contain blue T1 Cu sites. In addition, the Cys-His bridge contains two possible pathways for ET: a through-bond pathway (P1) and an H-bond pathway (P2). The difference between blue and green sites, as mentioned above, involves a different interaction between the Cu(3dx2-y2) orbital and the π and σ orbitals of the S(Cys) ligand, and thus the orbitals of the molecular bridge. Therefore, the electronic structure of the T1 site could also influence ET to the catalytic site. Chapter 7 focuses on the contribution of the Cu(II)-S(Cys) covalency and its anisotropy (i.e., π vs. σ) to the electronic coupling (HDA) between the donor and acceptor metal sites. The high covalency of the T1 Cu-S(Cys) bond activates the site for hole superexchange via occupied valence orbitals of the bridge through either P1 or P2 to the acceptor T2 Cu site. Besides the covalency of the donor, the covalency of the acceptor Cu site is also found to play an important role. This is covalency activated electronic coupling (HDA) and facilitates long-range ET. Furthermore, P1 and P2 of the Cys-His bridge can be selectively activated depending on the geometric and electronic structure of the T1 Cu site, where, in NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being overall more efficient. The higher efficiency of P2 results in a higher HDA for a green site relative to a blue site in NiR. By comparing the MCOs to NiRs, the second-sphere environment changes the geometric structure of the Cys-His pathway, which selectively activates HDA for efficient P2 superexchange by blue π sites, while it is deactivated for green σ sites, providing insight into why strictly blue T1 Cu sites are observed in the MCOs. Thus, in a biological architecture consisting of a T1 Cu donor/Cu-active site acceptor, the donor and acceptor geometric and electronic structures (i.e., the aniso ... .