Active Site Coordination Chemistry of the Cytochrome c Peroxidase Asp235Ala Variant: Spectroscopic and Functional Characterization

Abstract

Asp235 in yeast cytochrome c peroxidase forms a hydrogen bond with His175, the proximal histidyl residue, that has been suggested to be important in determining the electronic properties of the heme iron and that may be involved in stabilizing the higher oxidation states of the peroxidase that form transiently during catalysis. The current study employs 1H and 15N-NMR spectroscopy to study the electronic properties of and the effects of pH on the active site of the Asp235Ala variant. This variant exhibits three spectroscopic species between pH 5 and 9: a high-spin species that forms at low pH and two low-spin species that form successively at higher pH. Nevertheless, the activity of the variant exhibits a pH dependence virtually identical to that of the wild-type protein, though the activity of the variant is 3 orders of magnitude lower at all values of pH between pH 5 and 8.5. These findings suggest that the spin state and coordination environment of the heme iron in cytochrome c peroxidase do not dictate the rate of substrate (cytochrome c) oxidation. Binding of cyanide to the variant enzyme results in formation of a single species as detected by NMR spectroscopy. Analysis of high-resolution 1D and 2D 1H-NMR and 15N-NMR spectra of the cyanide adduct has permitted characterization of the properties of this derivative and the strength of the proximal ligand bond to the heme iron. Disruption of the hydrogen bond between the proximal histidine and Asp235 that exists in the wild-type enzyme dramatically reduces the strength of the interaction between the proximal ligand and the iron; this effect combined with concurrent changes in the distal heme-binding pocket accounts for the increase in reduction potential reported for the Fe3+/Fe2+ couple. The catalytic consequences of the structural and electronic properties of the variant elucidated in this study are discussed.

Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase

“…Recombinant wild-type yeast cytochrome-c peroxidase was obtained as previously described [9]. The sequence of this protein is identical to that of cytochrome-c peroxidase obtained from commercial yeast except for the three N-terminal residues, which have been changed from ThrThrPro to MetLysThr.…”

“…The sequence of this protein is identical to that of cytochrome-c peroxidase obtained from commercial yeast except for the three N-terminal residues, which have been changed from ThrThrPro to MetLysThr. The Arg484Glu mutation was introduced by site-directed mutagenesis following a procedure similar to that described for preparation of the Asp235dAla variant [9]. The sequence of the mutagenic oligonucleotide was 5'-P-CCCGTATTAGTCGAACTTG-CTTGGCAC-3'; the underlined triplet corresponds to the mutagenic codon.…”

“…1) to the heme iron coordination state and/or the activity of cytochrome-c peroxidase have been clearly established. For example, the hydrogen-bonding interactions of the Asp235/His175/Trp191 triad maintain the optimal orientation for formation of the radical on the Trpl91 side chain and provide a marked imidazolate character to the proximal ligand that is important in keeping the heme iron atom in the pentacoordinated state and in stabilizing compound I [9,13,141. Similarly, in the distal heme pocket, the hydrogen-bonding interactions of Trp51 participate in stabilizing the pentacoordinate state of the heme iron [6, 141. Of the other two important distal residues, His52 is the most critical for the rapid formation of compound I as illustrated by the observation that the His52tLeu variant exhibits a 105-fold decrease in the apparent second-order rate constant for the reaction with hydrogen peroxide [8,151.…”

Hemes férricos pentacoordenados e hexacoordenados dos monômeros d nativo e reconstituído da hemoglobina extracelular de Glossoscolex paulistus: estudos espectroscópicos em meio ácido

“…Assim sendo, ligantes que sejam bons σ-doadores, como por ex. água, imidazol e imidazolato, poderiam desestabilizar ligantes igualmente σ-doadores em sua posição trans, isto é, na sexta posição de coordenação 39,44,[49][50][51] . Por outro lado, Bertini e colaboradores 52 asseveram que a saída do centro férrico do plano porfirínico contribui estericamente para a estabilidade do pentacoordenado.…”

Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket

“…Also conserved is Asp#%#, which is located on the proximal side of the haem pocket, forming a hydrogen-bond with N δ of the imidazole group of the proximal His"($ residue [6]. There is an extensive debate in the literature about the role of this conserved hydrogen-bond in modulating the properties of the iron ion, and in particular its reduction potential, and consequently the reactivity of the protein [13][14][15][16]. An interesting structural aspect concerns the other residue located on the proximal side of the haem pocket near His"($, namely Phe"*!, as it displays some variation among different peroxidases.…”