Conformational Mobility in the Active Site of a Heme Peroxidase

Site-directed mutagenesis in recombinant soybean ascorbate peroxidase (rsAPX) has been performed to probe structure/function relationships in this and other heme proteins. In Chapter Two, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine (His-42) in the W41A variant of ascorbate peroxidase. In this variant, His-42 binds “on” to the heme in the oxidised form, duplicating the active site structure of the cytochromes b but, in contrast to cytochromes b, is able to swing “off” the iron during catalysis. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. In Chapter Three, we have shown that conformational rearrangement in W41A, discussed above, can also be triggered upon reduction of the heme iron. We present structural, spectroscopic and ligand binding data that support dissociation of His-42 from the iron in the ferrous form of W41A. Structural studies provide evidence for formation of a reduced, bis-histidine-ligated species that subsequently decays by dissociation of His-42 from the heme. Collectively, the data provide clear evidence that conformational movement within the same heme active site can be controlled by both ligand binding and metal oxidation state. In Chapter Four, we present evidence for heme oxygenase reactivity in the W41A variant of rsAPX. Crystallographic, spectroscopic, HPLC and MS techniques reveal that the heme is modified on reaction of W41A with tert-butyl hydroperoxide to yield a tert-butyl derivative of biliverdin. Evidence for formation of a hydroperoxo, Compound 0, intermediate is also presented. A common intermediate for the two heme enzymes is proposed. In Chapter Five, we examined the affect of disruption of the conserved distal histidine-asparagine hydrogen bond on active site mobility by formation of a series of asparagine-71 (Asn-71) variants. The spectroscopic data presented provide clear evidence that the conserved distal histidine-asparagine bond is required to maintain the correct orientation of distal histidine in the active site of rsAPX.