TY - JOUR
T1 - Partial conversion of Hansenula polymorpha amine oxidase into a "plant" amine oxidase
T2 - Implications for copper chemistry and mechanism
AU - Welford, Richard W.D.
AU - Lam, Angel
AU - Mirica, Liviu M.
AU - Klinman, Judith P.
PY - 2007/9/25
Y1 - 2007/9/25
N2 - The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O 2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O 2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/K m[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins.
AB - The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O 2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O 2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/K m[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins.
UR - http://www.scopus.com/inward/record.url?scp=34648825035&partnerID=8YFLogxK
U2 - 10.1021/bi700943r
DO - 10.1021/bi700943r
M3 - Article
C2 - 17760423
AN - SCOPUS:34648825035
SN - 0006-2960
VL - 46
SP - 10817
EP - 10827
JO - Biochemistry
JF - Biochemistry
IS - 38
ER -