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

Chelsey D. Kline, Ninian Blackburn

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 because 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.

Original languageEnglish (US)
Pages (from-to)6652-6661
Number of pages10
JournalBiochemistry
Volume55
Issue number48
DOIs
StatePublished - Dec 6 2016

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Carbon Monoxide
Conformations
Copper
Oxygen
Peptides
Substrates
Enzymes
Catalytic Domain
Chemical activation
Carbonylation
peptidylglycine monooxygenase
Electronic properties
Proteins
Stretching
Infrared radiation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase. / Kline, Chelsey D.; Blackburn, Ninian.

In: Biochemistry, Vol. 55, No. 48, 06.12.2016, p. 6652-6661.

Research output: Contribution to journalArticle

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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 because 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.",
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AB - 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 because 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.

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