Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties.
The gene coding for Pseudomonas aeruginosa cytochrome c-551 was expressed in Pseudomonas putida under aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosa showed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosa cytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the Fe-Met61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosa cytochrome c-551.
Cutruzzolà
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Istituto Pasteur-Fondazione Cenci-Bolognetti, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Italia.
Ciabatti
I
I
Rolli
G
G
Falcinelli
S
S
Arese
M
M
Ranghino
G
G
Anselmino
A
A
Zennaro
E
E
Silvestrini
M C
MC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D014364:Tryptophan; C017167:cytochrome C(551)