Abstract:The present study uses CO as a surrogate for oxygen to probe how substrate binding triggers oxygen activation in peptidylglycine monooygenase (PHM). Infrared stretching frequencies (ν(C≡O)) of the carbonyl (CO) adducts of copper proteins are sensitive markers of Cu(I) coordination, and are useful in probing oxygen reactivity since the electronic properties of O2 and CO are similar. The carbonyl chemistry has been explored using PHM WT and a number of active site variants in the absence and presence of peptidyl… Show more
“…In the wild-type enzyme, substrate binding has been shown to induce a new mode of CO binding at the M-center which lowers the C≡O infrared stretching frequency and suggests electronic activation of the diatomic ligand. This process does not occur in the mutants, consistent with a lack of substrate binding although clearly other factors could also be responsible 57 .…”
The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.
“…In the wild-type enzyme, substrate binding has been shown to induce a new mode of CO binding at the M-center which lowers the C≡O infrared stretching frequency and suggests electronic activation of the diatomic ligand. This process does not occur in the mutants, consistent with a lack of substrate binding although clearly other factors could also be responsible 57 .…”
The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.
“…The large DW for the enzyme has been suggested to arise from either two conformations at the M-center involving Met-on and Met-off forms (only one of which is active), or alternatively, scaffold-based protein dynamics that create a specific protein architecture and a fluxional Cu-S bond which might couple with other specialized vibrational modes involved in H-tunneling. 14,60,65 While these suggestions are elegant, the finding that the model also exhibits a large DW for Cu-S(met) at temperatures close to 10 K, establishes that this is a property of the ligand set, rather than a property induced by the PHM scaffold. Sufficient data exist to compare Cu-S(Met) DW values across a series of 3-coordinate CusF and CusB His x Met y ligand sets.…”
Section: Resultsmentioning
confidence: 99%
“…The trend in decreasing ν(CO) with increasing donor strength is exemplified by comparison of the WT PHM M-site carbonyl (2092 cm −1 ) with its M314H variant (2075 cm −1 ) where replacement of thioether with histidine results in a 17 cm −1 downshift. 60 Both CusF His 2 Met models bind CO within the His 2 Met ligand set range, with a v(CO) of 2086 and 2089 cm −1 for CusF M47H and M49H, respectively. The peak shape of both the M47H and M49H carbonyls was characterized with Gaussian peak fitting.…”
Section: Carbon Monoxide Bindingmentioning
confidence: 98%
“…FTIR data were recorded on a Bruker Tensor 27 FTIR spectrophotometer continuously purged with CO 2 -free dry air as previously described. 60 One thousand scans were collected for both protein sample and buffer blank from 2250 to 1900 cm −1 at a resolution of 2 cm −1 . Spectral analysis including subtraction of the buffer-blank was performed using GRAMS AI spectroscopy software (Thermo).…”
Section: Spectroscopic Data Collection and Processingmentioning
Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine βmonooxygenase (DBM) catalyze the hydroxylation of high energy C-H bonds utilizing a pair of chemically distinct copper sites (CuH and CuM) separated by 11 Å. In earlier work, we constructed single-site PHM variants that were designed to allow study of the M-and H-centers independently in order to place their reactivity sequentially along the catalytic pathway. More recent crystallographic studies suggest that these single-site variants may not be truly representative of the individual active sites. In this work we describe an alternative approach that uses rational design to construct an artificial PHM model in a small metallochaperone scaffold. Using site-directed mutagenesis, we constructed variants that provide a His 2 Met copper-binding ligand set that mimics the M-center of PHM. The results show that the model accurately reproduces the chemical and spectroscopic properties of the M-center, including details of the methionine coordination, and the properties of Cu(I) and Cu(II) states in the presence of endogenous ligands such as CO and azide. The rate of reduction of the Cu(II) form of the model by the chromophoric reductant N,N'-dimethyl phenylenediamine (DMPD) has been compared with that of the PHM M-center, and the reaction chemistry of the Cu(I) forms with molecular oxygen has also been explored, revealing an unusually low reactivity towards molecular oxygen. This latter finding emphasizes the importance of substrate triggering of oxygen reactivity, and implies that the His 2 Met ligand set while necessary, is insufficient on its own to activate oxygen in these enzyme systems.
“…O 2 activation and substrate hydroxylation occur at the Cu M site, while the Cu H site is responsible for electron transfer [57]. A very recent paper by Blackburn and Kline reports that a conformational change at the Cu M site occurs upon addition of substrate, postulated to promote O 2 binding and activation [58]. One major breakthrough was the structural characterization of dioxygen bound to Cu M (Fig.…”
Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing met-alloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O2-activation in copper proteins are addressed.
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