This CAREER award in the Inorganic, Bioinorganic and Organometallic Chemistry program and the Molecular and Cellular Biology program supports work by Professor Nicolai Lehnert at the University of Michigan to probe the interaction of nitric oxide (NO) with cytochrome P450. The goal of this project is to obtain fundamental insight into the spectrum of redox transformations of NO that can be catalyzed by cytochrome P450s. The investigations utilize both the enzyme cytochrome P450cam and synthetic model complexes. Magnetic circular dichroism and resonance Raman spectroscopy are used to establish the relative donor strengths of the thiolate ligands in different model complexes and P450cam wild type and mutants. Ferrous and ferric heme nitrosyls are prepared and their spectroscopic properties, electronic structures, and reactivities are studied as a function of the thiolate donor strength. Biologically relevant reactions of these nitrosyl complexes are then investigated, including in particular the reduction of NO to N2O as mediated by P450nor (NO detoxification) and the reaction of ferrous heme nitrosyls with O2 and superoxide (nitrosative stress). A new undergraduate course on the role of metals in life is being developed to attract underrepresented minority students to the basic sciences. The class focuses on the relation between fundamental biological and chemical processes and questions of high current impact in society like health, the environment and alternative energy sources. Follow-up programs for hands-on research experiences in these areas are designed for these students. Nitric oxide is a double-edged sword in biological systems: it is of central importance for many signaling and regulating processes, but at too high concentrations it also constitutes a deadly threat to the organism. The clarification of the fundamental reactions between cytochrome P450s and NO is of key importance for biological chemists to determine how P450s could participate in the biological network of NO signaling, transformation and detoxification. In particular, it is expected that the reactions between NO and cytochrome P450s identified in the research could potentially be relevant to human physiology.
Several gases released into the atmosphere by the activities of human civilization have been identified as green house gases that contribue to global warming. This includes carbon dioxide (CO2), methane (CH4) and nitrous oxide (laughing gas, N2O). The latter originates to a large degree from the over-fertilization of the farm land with inorganic nitrates. Denitrifying bacteria and fungi that live anaerobically in soil and sea water transform the nitrates into nitrous oxide in a large-scale process called dissimilatory denitrification, an anaerobic form of respiration, as shown in Image 1. Nitric oxide reductases (NORs) are the key enzymes responsible for the central transformation that generates N2O. In fungi, this process is mediated by Cyt. P450 NO reductase, Cyt. P450nor. Our mechanistic investigations on Cyt. P450nor, including the studies on models for the key intermediate in this enzyme, therefore contribute to the currently ongoing, large efforts to better understand N2O generation resulting from denitrification, and what the impact of this process is for global warming. Cyt. P450nor is particularly effective in catalyzing N2O formation, but it is currently unclear how the enzyme is able to mediate this reaction so efficiently. Another important contribution of this project to society relates to the importance of Cyt. P450s in medicine. Human macrophages employ a number of strategies to combat pathogenic infection, one being the release of nitric oxide (NO) at micromolar concentrations, which is very efficient in killing invading organisms. However, a number of pathogens have the ability to detoxify NO by reduction to N2O, using the above mentioned nitric oxide reductases (NORs). These enzymes are therefore capable of promoting pathogenic infection of the human body, leading to chronic infections and inflammations. As described above, NORs found in fungi belong to the cytochrome P450 enzyme family (Cyt. P450nor), which raises the question whether pathogens might also be able to use other Cyt. P450s as a platform for NO reduction and neutralization. Our work provides insight into this question by investigating the interaction of model complexes and Cyt. P450cam (and mutants) with NO, where Cyt. P450cam serves as a model for other Cyt. P450s found in bacteria. While decades of research have focused on the interaction of NO with hemoglobin/myoglobin, much less is known about the binding and redox transformations of nitric oxide with Cyt. P450s. This project has elucidated key elements of the mechanism of Cyt. P450nor, and how NO and Cyt P450s could interact, and how this could relate to human health. Finally, Cyt. P450s are also important model enzymes for the development of industrial green catalysts for the oxidation, hydroxylation, epoxidation, dealkylation, or degradation of organic compounds. Due to the environmentally detrimental effects of organic solvents, especially halogenated and aromatic compounds, there has been a major push for the usage of water in organic synthesis in the last 15 years. Elimination of organic solvents in chemical reactions is one of the core areas of Green Chemistry. Our research contributes to a better understanding of the significance of the proximal thiolate ligand and how the hydrogen bonding network around the cysteine (the ‘Cys’ pocket) fine-tunes the properties of the enzymatic catalyst. This information can be used to design improved biomimetic oxidation catalysts for industrial applications, which are based on the layout of the Cyt. P450 active site, and which could operate in water or alcohols as solvent. During the course of this project, a number of postdoctoral fellows, graduate students and undergraduate students were trained, a large percentage of them being females. Former postdoc Grace Galinato was able to obtain a faculty position at Penn State Erie as a result of the training that she has received. Other students moved on to successful positions in industry and academia (as postdoctoral fellow).
