Characterization of the tryptophan residues of Escherechia coli alkaline phosphatase by phosphorescence and optically detected magnetic resonance spectroscopy

The phosphorescence and zero field optically detected magnetic resonance (O DMR) of the tryptophan (Trp) residues of alkaline phosphatase from Escherec hia coli are examined. Each Trp is resolved optically and identified with t he aid of the W220Y mutant and the terbium complex of the apoenzyme. Trp(10 9), known from earlier work to be the source of room-temperature phosphores cence (RTP), emits a highly resolved low-temperature phosphorescence (LTP) spectrum and has the narrowest ODMR bands observed thus far from any protei n site, revealing a uniquely homogeneous local environment. The decay kinet ics of Trp(109) at 1.2 K reveals that the major triplet population (70%) un dergoes inefficient crystallike spin-lattice relaxation by direct interacti on with lattice phonons, the remainder being relaxed efficiently by local d isorder modes. The latter population is smaller than is typical for protein sites, suggesting an unusual degree of local rigidity and order consistent with the long-lived RTP. Trp(220) emits a broader LTP spectrum originating to the blue of Trp(109). It has typically broad ODMR bands consistent with local heterogeneity. The LTP of Trp(268) has an ill-defined origin blue sh ifted relative to Trp(220) and ODMR frequencies consistent with a greater d egree of solvent exposure. Trp268 has noticeable dispersion of its decay ki netics, consistent with quenching at the triplet level by a nearby disulfid e residue.