Conformational Mobility in the Active Site of a Heme Peroxidase

M Gordon Joyce,Peter C.E Moody,Jaswir Basran,Isabel K Macdonald,Sandip K Badyal,Katherine H Sharp,Emma Lloyd Raven,Martin Mewies,Harriet E Seward

doi:10.1074/jbc.m602602200

Abstract

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. 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. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

Highlights

Conformational Mobility in the Active Site of a Heme Peroxidase*We present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A)
Structural information is available for a number of heme peroxidase enzymes, and in all cases the heme iron is poised in a 5or 6-coordinate environment with the sixth ligand provided by a weakly coordinated water molecule
In line with the above considerations, there are no known examples of a genuine heme peroxidase with bis-histidine ligation, but there are a few examples in the literature of different heme peroxidases, or site-directed variants thereof, in which coordination of the distal histidine residue has been proposed on the basis of spectroscopic studies [3,4,5,6,7,8]

Summary

Conformational Mobility in the Active Site of a Heme Peroxidase*

We present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A) In this variant, His-42 binds “on” to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing “off” the iron during catalysis. We present the first crystallographically defined example of a functional peroxidase enzyme with bis-histidine ligation in the W41A variant of ascorbate peroxidase This variant duplicates the heme coordination geometry of the cytochromes b in the oxidized form but remains fully competent for formation of the catalytic Compound I and Compound II intermediates, as well as for sub-. These data indicate that strong coordination of the distal histidine residue to the heme iron does not automatically undermine peroxidase activity

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

PDB accession code

Structural Evidence for Mobility