The catalytic role of the copper ligand H172 of peptidylglycine α-hydroxylating monooxygenase (PHM)

A spectroscopic study of the H172A mutant

Shulamit Jaron, Richard E. Mains, Betty A. Eipper, Ninian Blackburn

Research output: Contribution to journalArticle

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Abstract

The spectroscopic characterization of the H172A mutant of peptidylglycine α-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this CuH ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 Å in wild-type PHM to 1.89 Å in H172A associated with a change in the structure of CuH from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the CuM site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at CuH was obtained through the observation that loss of the H172 CuH ligand caused a 3 cm-1 blue shift in the v(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.

Original languageEnglish (US)
Pages (from-to)13274-13282
Number of pages9
JournalBiochemistry
Volume41
Issue number44
DOIs
StatePublished - Nov 5 2002

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Carbon Monoxide
Mixed Function Oxygenases
Copper
Ligands
Binding Sites
Substrates
Enzymes
Histidine
Peptides
Mutation
peptidylglycine monooxygenase

ASJC Scopus subject areas

  • Biochemistry

Cite this

The catalytic role of the copper ligand H172 of peptidylglycine α-hydroxylating monooxygenase (PHM) : A spectroscopic study of the H172A mutant. / Jaron, Shulamit; Mains, Richard E.; Eipper, Betty A.; Blackburn, Ninian.

In: Biochemistry, Vol. 41, No. 44, 05.11.2002, p. 13274-13282.

Research output: Contribution to journalArticle

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abstract = "The spectroscopic characterization of the H172A mutant of peptidylglycine α-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this CuH ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 {\AA} in wild-type PHM to 1.89 {\AA} in H172A associated with a change in the structure of CuH from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the CuM site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at CuH was obtained through the observation that loss of the H172 CuH ligand caused a 3 cm-1 blue shift in the v(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.",
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AB - The spectroscopic characterization of the H172A mutant of peptidylglycine α-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this CuH ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 Å in wild-type PHM to 1.89 Å in H172A associated with a change in the structure of CuH from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the CuM site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at CuH was obtained through the observation that loss of the H172 CuH ligand caused a 3 cm-1 blue shift in the v(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.

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