Spectroscopic and Structural Analysis of Oxygen-Activating Nonheme Diiron Enzymes and Related Synthetic Models

University of Minnesota Ph.D. dissertation. May 2017. Major: Chemistry. Advisor: Lawrence Que. 1 computer file (PDF); xviii, 337 pages.The general mechanism of O2 activation by nonheme diiron enzymes begins when the diferrous iron cluster binds dioxygen. The diiron cluster is oxidized to a peroxo-diferric intermediate that in some cases reacts directly with substrates, and in others becomes further activated via the cleavage of the O–O bond, leading to the generation of a potent high-valent oxidant that is the active oxidant for the cycle. Peroxo-diferric intermediates are of high interest because they are crossroads between the use of peroxo-diferric or high-valent oxo intermediate as the active oxidant in diiron-cluster-mediated oxidase and oxygenase chemistry. Understanding this O2 activation process requires structural characterization of enzymatic peroxo-diferric species. Spectroscopic methods, like electronic absorbance, X-ray absorption (XAS), and resonance Raman (rR) spectroscopies are used to probe a rich landscape of oxygen-activated intermediates and obtain detailed structures of these species. Through systematic study, insight can be gained into the mechanisms of these biological systems and ultimately this insight can be used to understand how Nature has chosen to use peroxo-diferric intermediates for a variety of different functions. In Chapter 2, X-ray diffraction and XAS were used to characterize various form of the enzyme CmlA to understand how O2 is regulated in the presence and in the absence of its non-ribosomal peptide synthetase (NRPS) bound substrate. In Chapter 3, the intermediate species on the O2 activation pathway of the human enzyme deoxyhypusine hydroxylase (hDOHH), including the µ-1,2-peroxo species, were studied using XAS. The structural analysis of the active sites of the various hDOHH species provided insight into the reaction mechanism for the system. In Chapter 4, XAS and rR studies on the unusual peroxo-diferric species of the N-oxygenase CmlI were carried out. The spectroscopic analysis of the peroxo intermediate describes a new peroxo binding geometry for diiron enzymes, a µ-1,1-peroxo species. In Chapter 5, detailed XAS characterization of various synthetic peroxo-diferric and oxoiron(IV) model complexes is described. Overall, this thesis demonstrates the power of structural characterization by complementary spectroscopic methods to support and generate enzymatic mechanistic hypotheses

Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical

The Fe Protein: An Unsung Hero of Nitrogenase

Although the nitrogen-fixing enzyme nitrogenase critically requires both a reductase component (Fe protein) and a catalytic component, considerably more work has focused on the latter species. Properties of the catalytic component, which contains two highly complex metallocofactors and catalyzes the reduction of N2 into ammonia, understandably making it the “star” of nitrogenase. However, as its obligate redox partner, the Fe protein is a workhorse with multiple supporting roles in both cofactor maturation and catalysis. In particular, the nitrogenase Fe protein utilizes nucleotide binding and hydrolysis in concert with electron transfer to accomplish several tasks of critical importance. Aside from the ATP-coupled transfer of electrons to the catalytic component during substrate reduction, the Fe protein also functions in a maturase and insertase capacity to facilitate the biosynthesis of the two-catalytic component metallocofactors: fusion of the [Fe8S7] P-cluster and insertion of Mo and homocitrate to form the matured [(homocitrate)MoFe7S9C] M-cluster. These and key structural-functional relationships of the indispensable Fe protein and its complex with the catalytic component will be covered in this review

Dioxygen Activation by Nonheme Diiron Enzymes: Diverse Dioxygen Adducts, High-Valent Intermediates, and Related Model Complexes

A growing subset of metalloenzymes activates dioxygen with nonheme diiron active sites to effect substrate oxidations that range from the hydroxylation of methane and the desaturation of fatty acids to the deformylation of fatty aldehydes to produce alkanes and the six-electron oxidation of aminoarenes to nitroarenes in the biosynthesis of antibiotics. A common feature of their reaction mechanisms is the formation of O₂ adducts that evolve into more reactive derivatives such as diiron­(II,III)-superoxo, diiron­(III)-peroxo, diiron­(III,IV)-oxo, and diiron­(IV)-oxo species, which carry out particular substrate oxidation tasks. In this review, we survey the various enzymes belonging to this unique subset and the mechanisms by which substrate oxidation is carried out. We examine the nature of the reactive intermediates, as revealed by X-ray crystallography and the application of various spectroscopic methods and their associated reactivity. We also discuss the structural and electronic properties of the model complexes that have been found to mimic salient aspects of these enzyme active sites. Much has been learned in the past 25 years, but key questions remain to be answered

The Fe Protein: An Unsung Hero of Nitrogenase

Although the nitrogen-fixing enzyme nitrogenase critically requires both a reductase component (Fe protein) and a catalytic component, considerably more work has focused on the latter species. Properties of the catalytic component, which contains two highly complex metallocofactors and catalyzes the reduction of N2 into ammonia, understandably making it the “star” of nitrogenase. However, as its obligate redox partner, the Fe protein is a workhorse with multiple supporting roles in both cofactor maturation and catalysis. In particular, the nitrogenase Fe protein utilizes nucleotide binding and hydrolysis in concert with electron transfer to accomplish several tasks of critical importance. Aside from the ATP-coupled transfer of electrons to the catalytic component during substrate reduction, the Fe protein also functions in a maturase and insertase capacity to facilitate the biosynthesis of the two-catalytic component metallocofactors: fusion of the [Fe8S7] P-cluster and insertion of Mo and homocitrate to form the matured [(homocitrate)MoFe7S9C] M-cluster. These and key structural-functional relationships of the indispensable Fe protein and its complex with the catalytic component will be covered in this review

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N2. Nitrogenase is most commonly associated with the molybdenum-iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two "alternative nitrogenase" systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored

Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N2) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo-O5 (carboxyl) or Mo-O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process

Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli.

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the clean genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β‑Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor l-<i>p</i>-aminophenylalanine (l-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster that is reduced to the diferrous state (<b>WT</b>^<b>R</b>) to initiate O₂ activation. However, rapid O₂ activation occurs only when <b>WT</b>^<b>R</b> is bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of <b>WT</b>^<b>R</b> is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the <b>WT</b>^<b>R</b> cluster ligand structure as well as the structures of <b>WT</b>^<b>R</b> in complex with a functional CmlP variant (CmlP_AT) with and without l-PAPA attached. It is found that formation of the active <b>WT</b>^<b>R</b>:CmlP_AT–l-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of <b>WT</b>^<b>R</b> is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O₂ in the presence or absence of CmlP_AT–l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O₂ reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases