SITE-DIRECTED MUTAGENESIS OF MET243, A RESIDUE OF MYELOPEROXIDASE INVOLVED IN BINDING OF THE PROSTHETIC GROUP

The optical absorbance spectrum of reduced myeloperoxidase is red-shif ted with respect to that of other haemoproteins, and has the Soret ban d at 472 nm and the a band at 636 nm. The origin of the red shift is p oorly understood, but the interaction of the protein matrix with the c hromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzym e, and we have examined the effect of a Met243Gln mutation on the spec troscopic properties and catalytic activity of the enzyme. The mutatio n has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 4 72 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastica lly affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to per oxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a spec ial sulfonium linkage. The other factor which contributes is the prese nce of ester linkages between hydroxylated methyl groups on the haem a nd glutamate and aspartate residues, respectively. The results, combin ed with those of previous studies, now give us a comprehensive picture of the Various factors which contribute to the unusual optical proper ties of myeloperoxidase.