Probing the reactivity of different forms of azurin by flavin photoreduction

Abstract

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild‐type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5‐deazariboflavin and 8α‐imidazolyl‐(N‐propylyl)‐amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co‐ordination shell was filled with imidazole. 8α‐Imidazolyl‐(N‐propylyl)‐amino riboflavin appears one to two orders less efficient as a photo‐flash reductant than 5‐deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5‐fold lower than the WT reactivity (second‐order rate constants of 51 ± 2 × 107 m−1·s−1 versus 21 ± 1 × 107 m−1·s−1), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 107 m−1·s−1). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number . Structured digital abstract http://www.uniprot.org/uniprot/P00282 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/P00282 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8099460)

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin

“…S3). One is coordinated by His43 of one of the protomers, which also appears in other azurin structures . The other copper ion is coordinated by two N‐terminal amine groups (2.59, 2.67 Å), one backbone carbonyl (2.07 Å) and one backbone amide group (2.25 Å).…”

“…One is coordinated by His43 of one of the protomers, which also appears in other azurin structures. 45 The other copper ion is coordinated by two N-terminal amine groups (2.59, 2.67 Å ), one backbone carbonyl (2.07 Å ) and one backbone amide group (2.25 Å ). The two additional copper sites were formed due to the high copper concentration (0.1 mM) in the crystallization buffer.…”

Inner- and outer-sphere metal coordination in blue copper proteins

“…Canters and coworkers investigated azurin variants where His117 is replaced with glycine [79,80], which creates a “hole” in the azurin coordination sphere owing to a large rearrangement in the protein backbone [81]. The unsaturated coordination sphere allows for addition of exogenous ligands including halides, azide and imidazoles to the copper center.…”

“…From UV-visible (UV-vis), Raman and EPR data it was concluded that addition of imidazole “rescues” the T1 Cu center, while addition of anionic ligands enhances T2-character [79]. The open coordination site also allows ET and structural investigations using imidazole-modified complexes [81,82,83]. …”

Probing the Active Site of an Azurin Mutant Hot-Wired to Gold Electrodes

“…In this protein variant, the bulky Cu-coordinating residue His117 exposed on the protein surface (Figure a) has been replaced with the smaller residue Gly (both depicted in black), thus allowing for external access to the redox cavity (Figure b). In the absence of external ligands, the copper site contains one water molecule and is located 0.7 nm distance away from the protein surface . The other active site residues Gly 45, His 46, Cys 112, and Met 121 are shown in red.…”