Document Detail

HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.
MedLine Citation:
PMID:  23530865     Owner:  NLM     Status:  MEDLINE    
Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides 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 X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively 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 (electron paramagnetic resonance, XAS, and Fourier transform infrared) 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.
Chelsey D Kline; Mary Mayfield; Ninian J Blackburn
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-04-05
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-04-16     Completed Date:  2013-07-25     Revised Date:  2014-04-17    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2586-96     Citation Subset:  IM    
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MeSH Terms
Amino Acid Motifs
Catalytic Domain
Copper / metabolism*
Electron Spin Resonance Spectroscopy
Histidine / genetics
Hydrogen-Ion Concentration
Mixed Function Oxygenases / chemistry*,  genetics,  metabolism*
Models, Molecular
Multienzyme Complexes / chemistry*,  genetics,  metabolism*
Mutagenesis, Site-Directed
Oxygen / metabolism
Spectroscopy, Fourier Transform Infrared
Grant Support
Reg. No./Substance:
0/Multienzyme Complexes; 4QD397987E/Histidine; 789U1901C5/Copper; EC 1.-/Mixed Function Oxygenases; EC monooxygenase; S88TT14065/Oxygen

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