Water‐Based Hydrogen‐Atom Wires as Mediators in Long‐Range Proton‐Coupled Electron Transfer in Enzymes: A New Twist on Water Reactivity

Cárdenas, Diego J.; Cuerva, Juan M.; Alı́as, Miriam; Buñuel, Elena; Campaña, Araceli G.

doi:10.1002/chem.201100964

Cited by 22 publications

(25 citation statements)

References 73 publications

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“…A third suggestion from Klinman and coworkers (27) proposed that the electron could transfer across the intersite cavity, perhaps assisted by Y79 provided the solvent was ordered . This latter mechanism has also been tested in silico where a pathway composed of a chain of three H-bonded water molecules was calculated to have sufficiently low activation energy for rapid proton-coupled ET provided that the CuM-site acceptor was the Cu-O• (cupryl) species (61). A mechanism involving interdomain motion has also been suggested (39), but deemed unlikely on the grounds that the interdomain distance does not change in any of the crystal structures published to date.…”

Section: Discussionmentioning

confidence: 99%

“…The alternative pathway via His172 and the nearby Y79 which π-stacks against the histidine ring has been evaluated both experimentally (27, 41) and computationally (61). In this pathway, the tyrosine is about 7 Å from the CuM center, and could operate as a facile ET pathway provided the intervening solvent is ordered.…”

Section: Discussionmentioning

confidence: 99%

“…As the catalytic chemistry progresses an enzyme state is reached which is able to turn on the catalytic gate via H172 and Y79. It has been proposed that this event may be initiated by formation of a high potential intermediate such as a high-valent Cu(III)-oxo or Cu(II)-O •-species which is able to overcome the high reorganizational energy for ET through the H172-Y79 –water pathway (24, 61). One hypothesis worthy of merit posits that the electron “hops” from Y79 to the high-potential intermediate, forming a transient tyrosyl radical hole.…”

Section: Discussionmentioning

confidence: 99%

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Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

Chauhan

Hosseinzadeh

Lu³

et al. 2016

Biochemistry

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PHM is a dicopper enzyme that plays a vital role in the amidation of glycine extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies on the first electron transfer step (reductive phase) in the WT PHM as well as its variants. Stopped-flow was used to record the reduction kinetic traces using the chromophoric agent DMPD as the reductant. The reduction was found to be biphasic in the WT PHM with an initial fast phase (17.2s−1) followed by a much slower phase (0.46s−1). We were able to ascribe the fast and slow phase to the CuH and CuM-sites respectively by making use of the H242A and H107H108A mutants which only contain the CuH-site and CuM-site respectively. In the absence of substrate the redox potentials determined by cyclic voltammetry were 270 mV (CuH-site) and −15 mV (CuM-site), but binding of substrate (Ac-YVG) was found to alter both potentials so that they converged to a common value of 83 mV. Substrate binding also accelerated the slow reductive phase by ~10 fold, an effect that could be explained at least partially by the equalization of the reduction potential of the copper centers. Studies on H108A showed that the ET to the CuM-site is blocked, highlighting the role of the H108 ligand as a component of the reductive ET pathway. Strikingly, the rate of reduction of the H172A variant was unaffected despite the rate of catalysis being three orders of magnitude less than that of the WT PHM. These studies strongly indicate that the reductive phase and catalytic phase ET pathways are different and suggest a bifurcated ET pathway in PHM. We propose that H172 and Y79 form part of an alternate pathway for the catalytic phase ET while the H108 ligand along with the water molecules and substrate form the reductive phase ET pathway.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

Chauhan

Hosseinzadeh

Lu³

et al. 2016

Biochemistry

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“…Firstly the copper centers are 11 Å apart separated by a solvent-filled cleft in the protein which imposes restrictions on intra-site electron transfer (7-10). It has been proposed that the physical separation prevents the second electron from being transferred until after the enzyme has committed to catalysis, thereby harnessing the strong electrophilic reactivity of the Cu(II)-superoxo entity (11, 12).…”

Section: Introductionmentioning

confidence: 99%

Binding of Copper and Silver to Single-Site Variants of Peptidylglycine Monooxygenase Reveals the Structure and Chemistry of the Individual Metal Centers

et al. 2014

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Peptidylglycine monooxygenase (PHM) catalyzes the final step in the biosynthesis of amidated peptides that serve as important signaling molecules in numerous endocrine pathways. The catalytic mechanism has attracted much attention because of a number of unique attributes, including the presence of a pair of uncoupled copper centers separated by 11 Å (termed CuH and CuM), an unusual Cu(I)SMet interaction at the oxygen binding M-site, and the postulated Cu(II)–superoxo intermediate. Understanding the mechanism requires determining the catalytic roles of the individual copper centers and how they change during catalysis, a task made more difficult by the overlapping spectral signals from each copper center in the wild-type (WT) protein. To aid in this effort, we constructed and characterized two PHM variants that bound metal at only one site. The H242A variant bound copper at the H-center, while the H107AH108A double mutant bound copper at the M-center; both mutants were devoid of catalytic activity. Oxidized Cu(II) forms showed electron paramagnetic resonance and extended X-ray absorption fine structure (EXAFS) spectra consistent with their previously determined Cu(II)His3O and Cu(II)His2O2 ligand sets for the H- and M-centers, respectively. Cu(I) forms, on the other hand, showed unique chemistry. The M-center bound two histidines and a methionine at all pHs, while the H-center was two-coordinate at neutral pH but coordinated a new methionine S ligand at low pH. Fourier transform infrared studies confirmed and extended previous assignments of CO binding and showed unambiguously that the 2092 cm–1 absorbing species observed in the WT and many variant forms is an M-site Cu(I)–CO adduct. Silver binding was also investigated. When H107AH108A and M109I (a WT analogue with both sites intact) were incubated with excess AgNO3, each variant bound a single Ag(I) ion, from which it was inferred that Ag(I) binds selectively at the M-center with little or no affinity for the H-center. EXAFS at the Ag K-edge established a strong degree of similarity between the ligand sets of Cu and Ag bound at the M-center. These studies validate previous spectral assignments and provide new insights into the detailed chemistry of each metal site.

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“…Furthermore, crystallographic analysis reveals a structural interaction between the M and H sites, with the M314I inducing dissociation of the H107 ligand from the H-center, some 11 Å distant (6). H172 forms a stacking interaction with the conserved Y79 residue, and it has been suggested from studies on the related enzyme TBM (30), that the H172 ligand might form the exit pathway for the electron as it transfers from H to M using Y79 and oriented water molecules as additional elements of the ET pathway (31). …”

Section: Introductionmentioning

confidence: 99%

HHM Motif at the CuH-Site of Peptidylglycine Monooxygenase is a pH-Dependent Conformational Switch

2013

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Peptidylglycine monooxygenase is a copper-containing enzyme which catalyzes the amidation of neuropeptide hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108 and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8, and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the XAS. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819–10828) we tentitively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (EPR, XAS and FTIR) properties of a number of H-site variants, including H107A, H108A, H172A and M109I. Our results establish that M109 is indeed the coordinating ligand, and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general utility for the HHM motif as a copper- and pH-dependent conformational switch.

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Water‐Based Hydrogen‐Atom Wires as Mediators in Long‐Range Proton‐Coupled Electron Transfer in Enzymes: A New Twist on Water Reactivity

Cited by 22 publications

References 73 publications

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

Binding of Copper and Silver to Single-Site Variants of Peptidylglycine Monooxygenase Reveals the Structure and Chemistry of the Individual Metal Centers

HHM Motif at the CuH-Site of Peptidylglycine Monooxygenase is a pH-Dependent Conformational Switch

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