This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports work by Professor F. Ann Walker at the University of Arizona to probe the peroxidase activity of the nitrophorins (NPs). These ferriheme proteins, from the saliva of the blood-sucking insect Rhodnius prolixus, are known to sequester NO in the salivary glands and release it into the tissues of the victim during a bite. Preliminary findings have shown that these proteins also react with hydrogen peroxide or peroxyacetic acid (PAA) to oxidize typical substrates of peroxidase enzymes, as well as the important biological effector norepinephrine. The high-valent intermediate known as Compound I is formed after rapid mixing of H2O2 or PAA with NP. Two forms of Compound I are seen at different pH values; at high pH the typical Compound I spectrum is observed, while at low pH a different signal, novel for peroxidase enzymes, but previously seen in model heme complexes, is observed. The factors that cause the appearance of each of these signals, their disappearance to form tyrosyl radicals of the protein, and their subsequent reactivity with organic substrates, will be investigated in this project. A course on peroxidase and oxygenase enzymes will be developed and offered to undergraduate and graduate students in Chemistry, Biochemistry and Pharmaceutical Chemistry at the University of Arizona, and the course materials will be made available to others on the WWW. Peroxides and their oxidation reactions are important in chemical and biological systems and in industrial settings. The mechanisms of hydrogen peroxide (and organic peracid) reaction with transition metals are relevant to large scale chemistry and biochemistry, as well as to the reactions that produce deleterious Reactive Oxygen Species (ROS) in plants and animals. Investigation of the high valent reactivity of the novel ferriheme enzymes to be studied will provide insight into the factors that stabilize each of the high valent Compound I species and their subsequent reactivity with organic substrates.
This project involved a study of a set of proteins found in the saliva of a blood-sucking insect called nitrophorins. The primary purpose of these heme proteins is to store nitric oxide (NO) in the salivary glands at low pH for up to a month’s time and then to release the NO when the insect bites a victim and spits his saliva into the wound. As 5-coordinate heme proteins bound to histidine, and having the sixth coordination site available to bind NO or other ligands, we wondered whether these proteins could also serve as peroxidases, as do horseradish peroxidase (HRP) and a large number of other peroxidase enzymes, by reacting with hydrogen peroxide (H2O2) at the heme iron site to oxidize various substrates. We found that although the nitrophorins do not have charged residues near the heme to aid in the enzymatic mechanism, they indeed do oxidize various substrates including norepinephrine, a bioactive molecule known to constrict blood vessels. Nitrophorin 2 (NP2, 21% of the nitrophorin protein in the insect saliva, and the first nitrophorin to be expressed in the juvenile forms of the insect) was the major focus of this project, because it has the best spectroscopic properties of the four nitrophorins of the adult Rhodnius prolixus insect. Together with our collaborator, Dr. Annabella Ivancich, of the French Atomic Energy Commission in Saclay, near Paris, we investigated the peroxidase activity of NP2 by two major spectroscopic techniques, Electron Paramagnetic Resonance (EPR) and optical absorption spectroscopy, as shown in Fig. 3 and 4, respectively. We found that, when reacted with hydrogen peroxide or peroxyacetic acid, like HRP, NP2 also formed the high-valent iron state known as Compound I, which rapidly decayed by one electron to the state known as Compound II, simultaneously creating a protein radical. We speculated that the protein radical might be a tyrosine radical, and there are two tyrosines that are very close to the heme iron, as shown in Fig. 5. We therefore made three mutant proteins, where we mutated i) Tyr38 to Phe, ii) Tyr85 to Phe, and iii) both Tyr38 and Tyr85 to Phe. The EPR spectra of the mutants, after reaction with hydrogen peroxide, shown in Fig. 6, allowed us to conclude that Tyr38 (Y38) Is the site of the primary radical, while Tyr85 (Y85) is the site of the secondary radical if Tyr38 is not present. A major paper was published in Biochemistry in 2010 (49, 8857-8872), and two additional papers are in preparation for publication, with an additional one planned to be published in the future. One graduate student and two undergraduates were supported on this three-year grant, and one of the supported undergraduates acted as the teacher of five additional undergraduates (three senior biochemistry majors who were doing their Senior Capstone Research Projects in my laboratory, and two chemistry majors who had just completed their freshman year in college when they spent the summer of 2011 working in my lab as "volunteers". One of those two "volunteers" is doing her Molecular and Cellular Biology research in this laboratory during the 2012-13 academic year, while the other, a chemical engineering major, spent an exciting summer of 2012 working at Ventana Medical, Inc. here in Tucson. Both of the undergraduates supported on this grant have gone on to advanced training programs, one to graduate school and the other to a 2-year Physician’s Assistant training program.
