Probing the NO and CO sensing by chemotaxis periplasmic sensor domains from Geobacter sulfurreducens

ARIEL G. DE CANDIA, MARCELO A. MARTÍ, DANIEL H. MURGIDA

Salta

Encuentro; 3rd Latin American Protein Society Meeting ,; 2010

Latinamerican Protein Society

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in Geobacter sulfurreducens. They both possess a c-type heme group that “senses” a still unknown external stimulus. In order to address the NO and CO exogenous ligands as possible candidates, we undertook binding studies and spectroscopic characterization of the respective NO and CO adducts. The measured NO and CO dissociation constants of ferrous 582 and 935 sensors reveal high and similar affinities towards these small molecules. In the oxidized form, sensor 582 showed a much higher affinity for NO than the 935. Structural features of the CO and NO adducts were addressed by resonance Raman spectroscopy, on the basis of a detailed comparison of the ligand free and ligand bound proteins. Common to both sensors is that CO binds only to the ferrous form in the distal face of the heme, giving origin to six-coordinated low spin adducts by replacing Met60. On the other hand NO can bind to the heme in both ferrous and ferric states. In order to provide a structural and dynamical insight to the observed spectroscopic results we have performed 40 ns long MD simulations combined with QM/MM optimizations for each of both 582 and 935 homodimeric proteins in the different proposed oxidation/coordination states. These studies show that both the CO and NO bound structures do not present any specific ligand distal protein interaction and no significant difference is observed between them, suggesting that if ligand binding were the trigger it would be too unspecific.