Catalytic mechanism and protein engineering of copper-containing
nitrite reductase
Wijma, Hein Jakob
Citation
Wijma, H. J. (2006, February 9). Catalytic mechanism and protein engineering of
copper-containing nitrite reductase. Retrieved from https://hdl.handle.net/1887/4302
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Downloaded from:
https://hdl.handle.net/1887/4302
Catalytic Mechanism and Protein Engineering of
Copper-Containing Nitrite Reductase
Proefschrift
ter verkrij
ging
van de graad van Doctor aan de Universiteit Leiden,
op gezag van de Rector Magnificus Dr.
D.
D.
Breimer,
hoogleraar in de faculteit der W iskunde en
Natuurwetenschappen en die der Geneeskunde,
volgens besluit van het College voor Promoties
te verdedigen op donderdag 9 februari 2006
klokke 14:
15 uur
door
Hein Jakob W ij
ma
geboren te Smallingerland
Promotiecommissie
Promotor:
Prof. G.W. Canters
Copromotor:
Dr. M.P. Verbeet
Referent:
Prof. W.F. Hagen (Techni
sche Uni
versi
tei
t Del
ft)
Overi
ge l
eden: Prof. J. Brouwer
Cover:
Nitrite reductase from Alcaligenes faecalis S-6 with nitrite bound to the type-2 site
(1SJM). Shown are the residues that ligate the type-1 Cu atom (His95, Cys136, His 145,
and Met150), the residues that ligate the type-2 Cu atom (His100, His135, His306), and
residues that are involved in catalysis (Asp98 and His 255).
5
Contents
Abbreviations 6
1 Copper in BiologicalElectron Transfer 7 1.1 Electron Transfer in Nature and this Thesis 7 1.2 Structure and Spectral Properties of Type-1 and CuA Sites 10
1.3 Origin, Classification, and Function of Type-1 and CuA Site Containing Proteins 12
1.4 Electron Transfer Theory 15
1.5 Outline to this Investigation 20
2 Copper-containing Nitrite Reductase 21
2.1 General 21
2.2 Structure, Type-2 Site, and Related Active Sites 23
2.3 Diversity of NiR 24
2.4 Electron Donors and their Effect on Catalytic Activity 26
2.5 Effect of Ionic Strength on kcat 29
2.6 Catalytic Mechanism 30
2.7 Effect of Nitrite Concentration and pH on Catalytic Activity. 32 3 BidirectionalCatalysis by Copper-Containing Nitrite Reductase 35 4 A Random SequentialM echanism for Nitrite Binding and Active Site Reduction in
Copper-containing Nitrite Reductase
51 5 Catalytic Cycle of Copper-Containing Nitrite Reductase,Reversible Inactivation
and Irreversible Reduction
71 6 Reconstitution of the Type-1 Active Site of the H145G/A Variant of Nitrite
Reductase by Ligand Insertion
89 7 A Rearranging Ligand Enables Allosteric Controlof Catalytic Activity in
Copper-containing Nitrite Reductase
107 8 Reorganization Energy of the Type-1 Copper Site of Nitrite Reductase lowered by
its M ethionine Ligand
129
9 Conclusions,and Future Prospects 149
9.1 Catalysis by NiR 149
9.2 Engineering of External Ligands and Allosteric Effectors in Type-1 Copper Sites 151
