Structural and functional consequences of the substitution of proximal residues Cys-172 and Cys-192 in the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii
Résumé
Proximal cysteines 172 and 192 in the large subunit of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are evolutively conserved among cyanobacteria, algae, and higher plants. Mutation of Cys-172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here we report the effect of the substitution of Cys-172 and Cys-192 by serine on the catalytic properties, thermostability, and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor compared to the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V max} for carboxylation was similar to that of wild type Rubisco in the case of the C172S enzyme, but about 30% lower for the C192S Rubisco. In contrast, the K m} for CO 2} and O 2} were similar for C192S and wild-type enzymes, but distinctly higher (about double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature about 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side-chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.
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