Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.     

Substrate and metal control of barrier heights for oxo transfer to Mo and W bis-dithiolene sites

Reaction coordinates for oxo transfer from the substrates Me(3)NO, Me(2)SO, and Me(3)PO to the biologically relevant Mo(IV) bis-dithiolene complex [Mo(OMe)(mdt)(2)](-) where mdt = 1,2-dimethyl-ethene-1,2-dithiolate(2-), and from Me(2)SO to the analogous W(IV) complex, have been calculated using density functional theory. In each case, the reaction first proceeds through a transition state (TS1) to an intermediate with substrate weakly bound, followed by a second transition state (TS2) around which breaking of the substrate X-O bond begins. By analyzing the energetic contributions to each barrier, it is shown that the nature of the substrate and metal determines which transition state controls the rate-determining step of the reaction.     

Geometric and electronic structure of the heme-peroxo-copper complex [(F8TPP)FeIII-(O22-)-CuII(TMPA)](ClO4)

The geometric and electronic structure of the untethered heme-peroxo-copper model complex [(F(8)TPP)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](ClO(4)) (1) has been investigated using Cu and Fe K-edge EXAFS spectroscopy and density functional theory calculations in order to describe its geometric and electronic structure. The Fe and Cu K-edge EXAFS data were fit with a Cu...Fe distance of approximately 3.72 A. Spin-unrestricted DFT calculations for the S(T) = 2 spin state were performed on [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) as a model of 1. The peroxo unit is bound end-on to the copper, and side-on to the high-spin iron, for an overall mu-eta(1):eta(2) coordination mode. The calculated Cu...Fe distance is approximately 0.3 A longer than that observed experimentally. Reoptimization of [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) with a 3.7 A Cu...Fe constrained distance results in a similar energy and structure that retains the overall mu-eta(1):eta(2)-peroxo coordination mode. The primary bonding interaction between the copper and the peroxide involves electron donation into the half-occupied Cu d(z)2 orbital from the peroxide pi(sigma) orbital. In the case of the Fe(III)-peroxide eta(2) bond, the two major components arise from the donor interactions of the peroxide pi*(sigma) and pi*(v) orbitals with the Fe d(xz) and d(xy) orbitals, which give rise to sigma and delta bonds, respectively. The pi*(sigma) interaction with both the half-occupied d(z)2 orbital on the copper (eta(1)) and the d(xz) orbital on the iron (eta(2)), provides an effective superexchange pathway for strong antiferromagnetic coupling between the metal centers.     

Electron escape depth in silicon

The ESCA electron escape depth in silicon is determined from the peak areas in the electron spectra from evaporated thin films. For electron energies in the region 320 eV to 3.6 keV values from 13 to 83 Å are obtained. The escape depth in silicon dioxide is determined for the energies 1.6 and 3.6 keV. Binding energies and Auger energies are determined in silicon and silicon oxides.     

Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both L-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the Fe(II) site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).     

Spectroscopic Characterization of an Eight‐Iron Nitrogenase Cofactor Precursor that Lacks the “9th Sulfur”

Nitrogenases catalyze the reduction of N₂ to NH₄⁺ at its cofactor site. Designated the M‐cluster, this [MoFe₇S₉C(R‐homocitrate)] cofactor is synthesized via the transformation of a [Fe₄S₄] cluster pair into an [Fe₈S₉C] precursor (designated the L‐cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight‐iron cofactor precursor (designated the L*‐cluster), which is proposed to have the composition [Fe₈S₈C] and lack the “9^th sulfur” in the belt region of the L‐cluster. Our X‐ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*‐cluster represents a structural homologue to the l ‐cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*‐cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases.     

Fe(IV)═O(TBC)(CH3CN)]2+: comparative reactivity of iron(IV)-oxo species with constrained equatorial cyclam ligation

[Fe(IV)═O(TBC)(CH(3)CN)](2+) (TBC = 1,4,8,11-tetrabenzyl-1,4,8,11-tetraazacyclotetradecane) is characterized, and its reactivity differences relative to [Fe(IV)═O(TMC)(CH(3)CN)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) are evaluated in hydrogen atom (H-atom) abstraction and oxo-transfer reactions. Structural differences are defined using X-ray absorption spectroscopy and correlated to reactivities using density functional theory. The S = 1 ground states are highly similar and result in large activation barriers (~25 kcal/mol) due to steric interactions between the cyclam chelate and the substrate (e.g., ethylbenzene) associated with the equatorial π-attack required by this spin state. Conversely, H-atom abstraction reactivity on an S = 2 surface allows for a σ-attack with an axial substrate approach. This results in decreased steric interactions with the cyclam and a lower barrier (~9 kcal/mol). For [Fe(IV)═O(TBC)(CH(3)CN)](2+), the S = 2 excited state in the reactant is lower in energy and therefore more accessible at the transition state due to a weaker ligand field associated with the steric interactions of the benzyl substituents with the trans-axial ligand. This study is further extended to the oxo-transfer reaction, which is a two-electron process requiring both σ- and π-electron transfer and thus a nonlinear transition state. In oxo-transfer, the S = 2 has a lower barrier due to sequential vs concerted (S = 1) two electron transfer which gives a high-spin ferric intermediate at the transition state. The [Fe(IV)═O(TBC)(CH(3)CN)](2+) complex is more distorted at the transition state, with the iron farther out of the equatorial plane due to the steric interaction of the benzyl groups with the trans-axial ligand. This allows for better orbital overlap with the substrate, a lower barrier, and an increased rate of oxo-transfer.