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Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

ABSTRACT: The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75%, and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate of kBA ? (3.6 ps)(-1) is the dominant process, some CN(-) exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN(-) association rate, we find that the CN(-) escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ? (1-2) × 10(7) s(-1). This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1 × 10(7) s(-1)) under the same conditions. The analysis leads to an escape probability kout/(kout + kBA) ~ 10(-4), which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN(-) bimolecular association rate (170 M(-1) s(-1)), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN(-) bimolecular association rate is larger by ~10(3), making the CN(-) photolysis more difficult to observe.

SUBMITTER: Zeng W

PROVIDER: S-EPMC3631277 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

Zeng Weiqiao W Sun Yuhan Y Benabbas Abdelkrim A Champion Paul M PM

The journal of physical chemistry. B 20130403 15

The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75%, and the resonance Raman spectrum of the photoproduct ...[more]

PMID: 23472676

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Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Dataset Information

Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

Publications

Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

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