Conformation of pseudoazurin in the 152 kDa electron transfer complex with nitrite reductase determined by paramagnetic NMR.
Copper-containing nitrite reductase is able to catalyze the reduction of nitrite with a turnover rate of several hundreds per second. Electrons for the reaction are donated by the electron transfer protein pseudoazurin. The process of protein complex formation, electron transfer and dissociation must occur on the millisecond timescale to enable the fast turnover of the enzyme. The structure of this transient protein complex has been studied using paramagnetic NMR spectroscopy. Gadolinium complexes were attached specifically through two engineered Cys residues on three sites on the surface of nitrite reductase, causing strong distance-dependent relaxation effects on the residues of pseudoazurin. Docking of the two proteins based on these NMR-derived distance restraints and the chemical shift perturbation data shows convergence to a cluster of structures with an average root-mean-square deviation of 1.5 A. The binding interface consists of polar and non-polar residues surrounded by charges. The interprotein distance between the two type-1 copper sites is 15.5(+/-0.5) A, enabling fast interprotein electron transfer. The NMR-based lower limit estimate of 600 s(-1) for the dissociation rate constant and the fast electron transfer are consistent with the transient nature of the complex.
Vlasie
Monica D
MD
Leiden University, Institute of Chemistry, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Fernández-Busnadiego
Rubén
R
Prudêncio
Miguel
M
Ubbink
Marcellus
M
D001400:Azurin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004563:Electrochemistry; D004579:Electron Transport; D004583:Electrons; D005682:Gadolinium / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013056:Spectrophotometry, Ultraviolet; D013499:Surface Properties
D001426:Bacterial Proteins; C047163:pseudoazurin; D001400:Azurin; D005682:Gadolinium; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing; D003545:Cysteine