Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase

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 substrates. We have determined that upon carbonylation (i) a major CO band at 2092 cm−1 and a second minor CO band at 2063 cm−1 are observed in the absence of peptide substrate Ac-YVG; (ii) the presence of peptide substrate amplifies the minor CO band and causes it to partially interconvert with the CO band at 2092 cm−1; (iii) the substrate induced CO band is associated with a second conformer at CuM; and (iv) the CuH-site mutants which are inactive, fail to generate any substrate-induced CO bands. The total intensity of both bands is constant suggesting that the Cu(I)M site partitions between the two carbonylated enzyme states. Together, these data provide evidence for two conformers at CuM, one of which is induced by binding of the peptide substrate, with the implication that this represents the conformation that also allows binding and activation of O2.

Effects of copper occupancy on the conformational landscape of peptidylglycine α-hydroxylating monooxygenase

“…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 .

Fig. 6Interactions between Q170, Q272, H108, and two H 2 O molecules required to bind peptide substrate.

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Rational Design of a Histidine–Methionine Site Modeling the M-Center of Copper Monooxygenases in a Small Metallochaperone Scaffold

“…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.…”

“…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.…”

“…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).…”

Activation of dioxygen by copper metalloproteins and insights from model complexes

“…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.…”