B. Bhaskar

Crystal Structures of the Ferric, Ferrous, and Ferrous-NO Forms of the Asp140Ala Mutant of Human Heme Oxygenase-1: Catalytic Implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)-NO complex as a model for the Fe(II)-oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the alpha-meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.

A Novel Heme and Peroxide-Dependent Tryptophan-Tyrosine Cross-Link in a Mutant of Cytochrome c Peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.

Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase

We have previously shown that the K+ site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107–6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538–5545). All other peroxidases bind Ca2+ rather than K+. Using the K+-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca2+ selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na+, is bound instead of Ca2+. This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca2+ binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp191 radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca2+ more effectively than K+, demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.

Engineering Ascorbate Peroxidase Activity Into Cytochrome C Peroxidase

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.

Mesopone Cytochrome c Peroxidase: Functional Model of Heme Oxygenated Oxidases

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

Loop Stability in the Engineered Potassium Binding Site of Cytochrome c Peroxidase

Abstract The Trp 191 containing flexible loop of cytochrome c peroxidase (CcP) exists in equilibrium between open and closed conformers. The open conformer creates a cavity in the loop, which enables it to bind protonated forms of imidazole derivatives such 1,2-dimethylimidazolium (DMI). In the present study we have engineered the K + binding site into CcP and find the equilibrium of the conformer shifted in favor of the open form probably due to electrostatic destabilization. Subsequent changing of a hinge residue in the loop, Asn 195 , to Pro stabilizes the loop in the presence of the bound K + .