Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction.
Pseudoazurin (PAz), a well-characterized blue copper electron-transfer protein, is shown herein to be capable of mediating electron transfer to the nitrous oxide reductase (N(2)OR) from Achromobacter cycloclastes (Ac). Spectroscopic measurements demonstrate that reduced PAz is efficiently re-oxidized by a catalytic amount of N(2)OR in the presence of N(2)O. Fits of the kinetics resulted in K(M) (N(2)O) and k(cat) values of 19.1±3.8 μM and 89.3±4.2s(-1) respectively. The K(M) (PAz) was 28.8±6.6 μM. The electrochemistry of Ac pseudoazurin (AcPAz) in the presence of Ac nitrous oxide reductase (AcN(2)OR) and N(2)O displayed an enhanced cathodic sigmoidal current-potential curve, in excellent agreement with the re-oxidation of reduced AcPAz during the catalytic reduction of N(2)O by AcN(2)OR. Modeling the structure of the AcPAz-AcN(2)OR electron transfer complex indicates that AcPAz binds near Cu(A) in AcN(2)OR, with parameters consistent with the formation of a transient, weakly-bound complex. Multiple, potentially efficient electron-transfer pathways between the blue-copper center in AcPAz and Cu(A) were also identified. Collectively, the data establish that PAz is capable of donating electrons to N(2)OR in N(2)O reduction and is a strong candidate for the physiological electron donor to N(2)OR in Ac.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA. fujitak@med.umich.edu
Koyu
K
Fujita
Mika
M
Hirasawa-Fujita
Doreen E
DE
Brown
Yuji
Y
Obara
Fumihiro
F
Ijima
Takamitsu
T
Kohzuma
David M
DM
Dooley
D042422:Achromobacter cycloclastes / Q000737:chemistry*; D001400:Azurin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004579:Electron Transport; D007700:Kinetics; D009609:Nitrous Oxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*
D001426:Bacterial Proteins; C047163:pseudoazurin; D001400:Azurin; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D009609:Nitrous Oxide