TY - JOUR
T1 - Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase
AU - Chauhan, Shefali
AU - Hosseinzadeh, Parisa
AU - Lu, Yi
AU - Blackburn, Ninian J.
N1 - Funding Information:
The work was funded by National Institutes of Health Grant R01 GM GM115214 (N.J.B.) and by National Science Foundation Grant CHE 14-13328 (Y.L.). Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393).
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/4/19
Y1 - 2016/4/19
N2 - Peptidylglycine monooxygenase (PHM) is a dicopper enzyme that plays a vital role in the amidation of glycine-extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and -transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies of the first electron transfer (ET) step (reductive phase) in wild-type (WT) PHM as well as its variants. Stopped flow was used to record the reduction kinetic traces using the chromophoric agent N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) as the reductant. The reduction was found to be biphasic in the WT PHM with an initial fast phase (17.2 s-1) followed by a much slower phase (0.46 s-1). We were able to ascribe the fast and slow phase to the CuH and CuM sites, respectively, by making use of the H242A and H107AH108A mutants that contain only the CuH site and CuM site, respectively. In the absence of substrate, the redox potentials determined by cyclic voltammetry were 270 mV (CuH site) and -15 mV (CuM site), but binding of substrate (Ac-YVG) was found to alter both potentials so that they converged to a common value of 83 mV. Substrate binding also accelerated the slow reductive phase by ∼10-fold, an effect that could be explained at least partially by the equalization of the reduction potential of the copper centers. Studies of H108A showed that the ET to the CuM site is blocked, highlighting the role of the H108 ligand as a component of the reductive ET pathway. Strikingly, the rate of reduction of the H172A variant was unaffected despite the rate of catalysis being 3 orders of magnitude slower than that of the WT PHM. These studies strongly indicate that the reductive phase and catalytic phase ET pathways are different and suggest a bifurcated ET pathway in PHM. We propose that H172 and Y79 form part of an alternate pathway for the catalytic phase ET while the H108 ligand along with the water molecules and substrate form the reductive phase ET pathway.
AB - Peptidylglycine monooxygenase (PHM) is a dicopper enzyme that plays a vital role in the amidation of glycine-extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and -transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies of the first electron transfer (ET) step (reductive phase) in wild-type (WT) PHM as well as its variants. Stopped flow was used to record the reduction kinetic traces using the chromophoric agent N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) as the reductant. The reduction was found to be biphasic in the WT PHM with an initial fast phase (17.2 s-1) followed by a much slower phase (0.46 s-1). We were able to ascribe the fast and slow phase to the CuH and CuM sites, respectively, by making use of the H242A and H107AH108A mutants that contain only the CuH site and CuM site, respectively. In the absence of substrate, the redox potentials determined by cyclic voltammetry were 270 mV (CuH site) and -15 mV (CuM site), but binding of substrate (Ac-YVG) was found to alter both potentials so that they converged to a common value of 83 mV. Substrate binding also accelerated the slow reductive phase by ∼10-fold, an effect that could be explained at least partially by the equalization of the reduction potential of the copper centers. Studies of H108A showed that the ET to the CuM site is blocked, highlighting the role of the H108 ligand as a component of the reductive ET pathway. Strikingly, the rate of reduction of the H172A variant was unaffected despite the rate of catalysis being 3 orders of magnitude slower than that of the WT PHM. These studies strongly indicate that the reductive phase and catalytic phase ET pathways are different and suggest a bifurcated ET pathway in PHM. We propose that H172 and Y79 form part of an alternate pathway for the catalytic phase ET while the H108 ligand along with the water molecules and substrate form the reductive phase ET pathway.
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U2 - 10.1021/acs.biochem.6b00061
DO - 10.1021/acs.biochem.6b00061
M3 - Article
C2 - 26982589
AN - SCOPUS:84964319921
SN - 0006-2960
VL - 55
SP - 2008
EP - 2021
JO - Biochemistry
JF - Biochemistry
IS - 13
ER -