Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

Abstract

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagmetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.