1DSP

CYTOCHROME C PEROXIDASE H175G MUTANT, IMIDAZOLE COMPLEX AT PH 7, ROOM TEMPERATURE.

Summary for 1DSP

• Entry DOI: 10.2210/pdb1dsp/pdb
• Related: 1CCA 1DS4 1DSE 1DSG 1DSO
• Descriptor: CYTOCHROME C PEROXIDASE, PROTOPORPHYRIN IX CONTAINING FE, IMIDAZOLE, ... (4 entities in total)
• Functional Keywords: heme enzyme, peroxidase, cavity mutant, ligand binding, oxidoreductase
• Biological source: Saccharomyces cerevisiae (baker's yeast)
• Cellular location: Mitochondrion matrix: P00431
• Total number of polymer chains: 1
• Total formula weight: 33931.52

Authors

Hirst, J.,Wilcox, S.K.,Williams, P.A.,McRee, D.E.,Goodin, D.B. (deposition date: 2000-01-07, release date: 2001-03-07, Last modification date: 2024-02-07)

Primary citation

Hirst, J.,Wilcox, S.K.,Williams, P.A.,Blankenship, J.,McRee, D.E.,Goodin, D.B.
Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure.
Biochemistry, 40:1265-1273, 2001

PubMed Abstract: Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.

PubMed: 11170452
DOI: 10.1021/bi002089r

Experimental method

X-RAY DIFFRACTION (2.03 Å)