Synthesis and Spectroscopic Characterization of Ferric Heme-Thiolate Complexes and Their Reactivity with NO as Models for Cytochrome P450 Nitric Oxide Reductase

Abstract

Nitric oxide (NO) is a key intermediate in the global nitrogen cycle, especially in microbial denitrification. For example, NO is produced by copper nitrite reductases (CuNIR) and in fungi, its reduction to nitrous oxide (N2O) is mediated by cytochrome (Cyt) P450 NO reductase (P450nor). Analogous to other Cyt P450 enzymes, key features of the P450nor active site include a heme b center with a bound proximal cysteinate (a thiolate ligand) and the Cys-pocket, which forms hydrogen bonds with the proximal thiolate sulfur. In the first step of the P450nor catalytic cycle, the ferric resting state binds NO to generate a ferric heme-nitrosyl, or {FeNO}6 (in the Enemark-Feltham notation), intermediate. However, the exact electronic effect of the thiolate donor on the properties of the Fe-N-O unit have remained elusive, in part due to the lack of proper model systems to study this effect systematically. In this work, I present the low temperature preparation of eleven new heme-thiolate {FeNO}6 model complexes, [Fe(TPP)(SPh*)(NO)] and [Fe(TPP)(SPh-NHPh-pR)(NO)], and their characterization by UV-Vis, IR, and resonance Raman spectroscopy. These eleven heme-thiolate {FeNO}6 complexes utilize two unique series of thiolate ligands, six ‘electron-poor’ thiophenolates (SPh*−) and five thiophenolates containing an intramolecular hydrogen bond (SPh-NHPh-pR−). Through their characterization, this work has now experimentally demonstrated that there is a direct correlation between the thiolate donor strength and the Fe-NO and N-O bond strengths due to a thiolate sigma-trans effect that can be spectroscopically measured by determination of the Fe-NO and N-O stretching frequencies. Furthermore, it is demonstrated that the strength of the proximal hydrogen bonds to the thiolate can be gauged by determination of the vibrational properties of the Fe-N-O unit. Besides their electronic effect, it is demonstrated here that these hydrogen bonds have a protective function for the thiolate ligand against S-nitrosylation. Through the use of DFT calculations, the thiolate sigma-trans effect is also found to be the electronic origin responsible for the “push effect”, which is proposed to accelerate O-O bond cleavage and Compound I formation in Cyt P450 monooxygenase catalysis. Thus, it is shown that the Fe-NO and N-O vibrational frequencies of heme-thiolate {FeNO}6 complexes can be used as a sensitive probe to quantify the thiolate donor strength of heme-thiolate complexes and the magnitude of the “push effect” in P450 monooxygenase enzymes.

Additionally, the synthesis and characterization of six Cu(II) BMPA- and BEPA-carboxylate complexes (BMPA = bis-(2-methylpyridyl)amine); BEPA = bis-(2-ethylpyridyl) amine) with varied carboxylate chain lengths, and the detailed electrochemical testing of their ability to reduce nitrite (NO2̄ ) to NO in aqueous media at pH 7.4, is presented. The redox potentials of these six complexes are found to span approximately 400 mV, which has allowed us to systematically determine the effect of the reduction potential of the Cu(II) complexes on the observed Faradaic efficiencies for electrocatalytic NO2̄ reduction to NO. It is concluded that the Cu(I) complexes in this series with more positive reduction potentials are less active in further reduction/disproportionation of the generated NO to N2O, and are thus more selective for electrocatalytic NO generation.