Heather R. Williamson

Tryptophan-accelerated electron flow across a protein-protein interface.

We report a new metallolabeled blue copper protein, Re126W122Cu(I) Pseudomonas aeruginosa azurin, which has three redox sites at well-defined distances in the protein fold: Re(I)(CO)3(4,7-dimethyl-1,10-phenanthroline) covalently bound at H126, a Cu center, and an indole side chain W122 situated between the Re and Cu sites (Re-W122(indole) = 13.1 Å, dmp-W122(indole) = 10.0 Å, Re-Cu = 25.6 Å). Near-UV excitation of the Re chromophore leads to prompt Cu(I) oxidation (<50 ns), followed by slow back ET to regenerate Cu(I) and ground-state Re(I) with biexponential kinetics, 220 ns and 6 μs. From spectroscopic measurements of kinetics and relative ET yields at different concentrations, it is likely that the photoinduced ET reactions occur in protein dimers, (Re126W122Cu(I))2 and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: *Re//←W122←Cu(I), where // denotes a protein-protein interface. Solution mass spectrometry confirms a broad oligomer distribution with prevalent monomers and dimers, and the crystal structure of the Cu(II) form shows two Re126W122Cu(II) molecules oriented such that redox cofactors Re(dmp) and W122-indole on different protein molecules are located at the interface at much shorter intermolecular distances (Re-W122(indole) = 6.9 Å, dmp-W122(indole) = 3.5 Å, and Re-Cu = 14.0 Å) than within single protein folds. Whereas forward ET is accelerated by hopping through W122, BET is retarded by a space jump at the interface that lacks specific interactions or water molecules. These findings on interfacial electron hopping in (Re126W122Cu(I))2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.

Oxidative damage in MauG: implications for the control of high-valent iron species and radical propagation pathways.

The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H2O2 with the diferric form of MauG, or reaction of O2 with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is more than 40 Å from the high-spin heme iron where H2O2 initially reacts, and catalysis relies on radical hopping through an interfacial residue, Trp199 of MauG. In the absence of preMADH, the bis-Fe(IV) intermediate is remarkably stable, but repeated exposure to H2O2 results in suicide inactivation. Using mass spectrometry, we show that this process involves the oxidation of three Met residues (108, 114, and 116) near the high-spin heme through ancillary electron transfer pathways engaged in the absence of substrate. The mutation of a conserved Pro107 in the distal pocket of the high-spin heme results in a dramatic increase in the level of oxidation of these Met residues. These results illustrate structural mechanisms by which MauG controls reaction with its high-valent heme cofactor and limits uncontrolled oxidation of protein residues and loss of catalytic activity. The conservation of Met residues near the high-spin heme among MauG homologues from different organisms suggests that eventual deactivation of MauG may function in a biological context. That is, methionine oxidation may represent a protective mechanism that prevents the generation of reactive oxygen species by MauG in the absence of preMADH.

Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis-FeIV state of MauG

Significance The diheme enzyme MauG stabilizes a unique high-valent redox state in which the two hemes are each present as FeIV and communicate via an intervening tryptophan residue. A network of amino acid side chains and structured waters are used to coordinate multiple proton and electron transfers that are responsible for the charge–resonance stabilization of the high-valent state and control of its reactivity. This describes a rare example of how an enzyme can use multiple pathways involving multiple-proton transfers in the transition state of a reaction, and demonstrates what may be a previously unrecognized mechanism by which proteins are able to control and use otherwise highly reactive transient species. The high-valent state of the diheme enzyme MauG exhibits charge–resonance (CR) stabilization in which the major species is a bis-FeIV state with one heme present as FeIV=O and the other as FeIV with axial heme ligands provided by His and Tyr side chains. In the absence of its substrate, the high-valent state is relatively stable and returns to the diferric state over several minutes. It is shown that this process occurs in two phases. The first phase is redistribution of the resonance species that support the CR. The second phase is the loss of CR and reduction to the diferric state. Thermodynamic analysis revealed that the rates of the two phases exhibited different temperature dependencies and activation energies of 8.9 and 19.6 kcal/mol. The two phases exhibited kinetic solvent isotope effects of 2.5 and 2.3. Proton inventory plots of each reaction phase exhibited extreme curvature that could not be fit to models for one- or multiple-proton transfers in the transition state. Each did fit well to a model for two alternative pathways for proton transfer, each involving multiple protons. In each case the experimentally determined fractionation factors were consistent with one of the pathways involving tunneling. The percent of the reaction that involved the tunneling pathway differed for the two reaction phases. Using the crystal structure of MauG it was possible to propose proton–transfer pathways consistent with the experimental data using water molecules and amino acid side chains in the distal pocket of the high-spin heme.

Mechanism of protein oxidative damage that is coupled to long-range electron transfer to high-valent haems.

In the absence of its substrate, the auto-reduction of the high-valent bis-Fe(IV) state of the dihaem enzyme MauG is coupled to oxidative damage of a methionine residue. Transient kinetic and solvent isotope effect studies reveal that this process occurs via two sequential long-range electron transfer (ET) reactions from methionine to the haems. The first ET is coupled to proton transfer (PT) to the haems from solvent via an ordered water network. The second ET is coupled to PT at the methionine site and occurs during the oxidation of the methionine to a sulfoxide. This process proceeds via Compound I- and Compound II-like haem intermediates. It is proposed that the methionine radical is stabilized by a two-centre three-electron (2c3e) bond. This provides insight into how oxidative damage to proteins may occur without direct contact with a reactive oxygen species, and how that damage can be propagated through the protein.

Mechanisms for control of biological electron transfer reactions.

Electron transfer (ET) through and between proteins is a fundamental biological process. The rates and mechanisms of these ET reactions are controlled by the proteins in which the redox centers that donate and accept electrons reside. The protein influences the magnitudes of the ET parameters, the electronic coupling and reorganization energy that are associated with the ET reaction. The protein can regulate the rates of the ET reaction by requiring reaction steps to optimize the system for ET, leading to kinetic mechanisms of gated or coupled ET. Amino acid residues in the segment of the protein through which long range ET occurs can also modulate the ET rate by serving as staging points for hopping mechanisms of ET. Specific examples are presented to illustrate these mechanisms by which proteins control rates of ET reactions.

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

GoxA is a glycine oxidase bearing a protein-derived cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications catalyzed by a flavoprotein, GoxB. Two forms of GoxA were isolated: an active form with mature CTQ and an inactive precursor protein that lacked CTQ. The active GoxA was present as a homodimer with no detectable affinity for GoxB, whereas the precursor was isolated as a monomer in a tight complex with one GoxB. Thus, the interaction of GoxA with GoxB and subunit assembly of mature GoxA are each dependent on the extent of CTQ biosynthesis.

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

Mass Spectrometric Characterization of Oligomers in Pseudomonas aeruginosa Azurin Solutions

We have employed laser-induced liquid bead ion desorption mass spectroscopy (LILBID MS) to study the solution behavior of Pseudomonas aeruginosa azurin as well as two mutants and corresponding Re-labeled derivatives containing a Re(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(+) chromophore appended to a surface histidine. LILBID spectra show broad oligomer distributions whose particular patterns depend on the solution composition (pure H(2)O, 20-30 mM NaCl, 20 and 50 mM NaP(i) or NH(4)P(i) at pH = 7). The distribution maximum shifts to smaller oligomers upon decreasing the azurin concentration and increasing the buffer concentration. Oligomerization is less extensive for native azurin than its mutants. The oligomerization propensities of unlabeled and Re-labeled proteins are generally comparable, and only Re126 shows some preference for the dimer that persists even in highly diluted solutions. Peak shifts to higher masses and broadening in 20-50 mM NaP(i) confirm strong azurin association with buffer ions and solvation. We have found that LILBID MS reveals the solution behavior of weakly bound nonspecific protein oligomers, clearly distinguishing individual components of the oligomer distribution. Independently, average data on oligomerization and the dependence on solution composition were obtained by time-resolved anisotropy of the Re-label photoluminescence that confirmed relatively long rotation correlation times, 6-30 ns, depending on Re-azurin and solution composition. Labeling proteins with Re-chromophores that have long-lived phosphorescence extends the time scale of anisotropy measurements to hundreds of nanoseconds, thereby opening the way for investigations of large oligomers with long rotation times.

Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis.

GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2. This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcat and Km values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.

A T67A mutation in the proximal pocket of the high-spin heme of MauG stabilizes formation of a mixed-valent FeII/FeIII state and enhances charge resonance stabilization of the bis-FeIV state

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket.