Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins.
The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.
Kanbi
Lalji D
LD
Faculty of Applied Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
Antonyuk
Svetlana
S
Hough
Michael A
MA
Hall
John F
JF
Dodd
Fraser E
FE
Hasnain
S Samar
SS
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry*; D001665:Binding Sites; D002570:Ceruloplasmin / Q000737:chemistry; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D042845:Laccase; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D010802:Phylogeny; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D013855:Thiobacillus / Q000378:metabolism*
D008024:Ligands; C050107:cupredoxin; C012543:rusticyanin; D001400:Azurin; D001224:Aspartic Acid; D012694:Serine; D003300:Copper; D008715:Methionine; D010088:Oxidoreductases; D042845:Laccase; D002570:Ceruloplasmin; D009572:Nitrite Reductases; D007930:Leucine