Erik T. Yukl

Transcription factor NsrR from Bacillus subtilis senses nitric oxide with a 4Fe-4S cluster

In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here, we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S](2+) cluster, which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O(2). These results, together with the fact that there are only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a noncysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and cross-linking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S](2+) holo form as well as in the apo form. These findings provide a first stepping stone to investigate the mechanism of NO sensing in NsrR.

Mutagenesis of tryptophan199 suggests that hopping is required for MauG-dependent tryptophan tryptophylquinone biosynthesis

The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of preMADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with preMADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with kcat/Km values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.

Kinetic and Spectroscopic Studies of Hemin Acquisition in the Hemophore HasAp from Pseudomonas aeruginosa

The extreme limitation of free iron has driven various pathogens to acquire iron from the host in the form of heme. Specifically, several Gram-negative pathogens secrete a heme binding protein known as HasA to scavenge heme from the extracellular environment and to transfer it to the receptor protein HasR for import into the bacterial cell. Structures of heme-bound and apo-HasA homologues show that the heme iron(III) ligands, His32 and Tyr75, reside on loops extending from the core of the protein and that a significant conformational change must occur at the His32 loop upon heme binding. Here, we investigate the kinetics of heme acquisition by HasA from Pseudomonas aeruginosa (HasAp). The rate of heme acquisition from human met-hemoglobin (met-Hb) closely matches that of heme dissociation which suggests a passive mode of heme uptake from this source. The binding of free hemin is characterized by an initial rapid phase forming an intermediate before further conversion to the final complex. Analysis of this same reaction using an H32A variant lacking the His heme ligand shows only the rapid phase to form a heme-protein complex spectroscopically equivalent to that of the wild-type intermediate. Further characterization of these reactions using electron paramagnetic resonance and resonance Raman spectroscopy of rapid freeze quench samples provides support for a model in which heme is initially bound by the Tyr75 to form a high-spin heme-protein complex before slower coordination of the His32 ligand upon closing of the His loop over the heme. The slow rate of this loop closure implies that the induced-fit mechanism of heme uptake in HasAp is not based on a rapid sampling of the H32 loop between open and closed configurations but, rather, that the H32 loop motions are triggered by the formation of the high-spin heme-HasAp intermediate complex.

The millisecond intermediate in the reaction of nitric oxide with oxymyoglobin is an iron(III)--nitrato complex, not a peroxynitrite.

The dioxygenation of nitric oxide by oxyheme in globin proteins is a major route for NO detoxification in aerobic biological systems. In myoglobin, this reaction is thought to proceed through an iron(III)-bound peroxynitrite before homolytic cleavage of the O-O bond to form an iron(IV)-oxo and NO(2) radical followed by recombination and nitrate production. Single turnover experiments at alkaline pH have revealed the presence of a millisecond high-spin heme intermediate. It is widely presumed that this species is an iron(III)-peroxynitrite species, but detailed characterization of the intermediate is lacking. Using resonance Raman spectroscopy and rapid-freeze quench techniques, we identify the millisecond intermediate as an iron(III)-nitrato complex with a symmetric NO(2) stretch at 1282 cm(-1). Greater time resolution techniques will be required to detect the putative iron(III) peroxynitrite complex.

The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H2O2, the α-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When 18O-labeled H2O2 was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with 18O and must therefore be derived from H2O2. However, when the reaction was carried out with H 162O2 in the presence of 18O2, 60% of the product contained the 18O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual six-electron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.

Nitric oxide‐sensitive and ‐insensitive interaction of Bacillus subtilis NsrR with a ResDE‐controlled promoter

NsrR is a nitric oxide (NO)‐sensitive transcription repressor that controls NO metabolism in a wide range of bacteria. In Bacillus subtilis, NsrR represses transcription of the nitrite reductase (nasDEF) genes that are under positive control of the ResD–ResE two‐component signal transduction system. Derepression is achieved by reaction of NO with NsrR. Unlike some NsrR orthologues that were shown to contain a NO‐sensitive [2Fe–2S] cluster, B. subtilis NsrR, when purified anaerobically either from aerobic or from anaerobic Escherichia coli and B. subtilis cultures, contains a [4Fe–4S] cluster. [4Fe–4S]‐NsrR binds around the −35 element of the nasD promoter with much higher affinity than apo‐NsrR and binding of [4Fe–4S]‐NsrR, but not apo‐protein, is sensitive to NO. RNA polymerase and phosphorylated ResD make a ternary complex at the nasD promoter and NsrR dissociates the preformed ternary complex. In addition to the −35 region, NsrR binds to two distinct sites of the upstream regulatory region where ResD also binds. These interactions, unlike the high‐affinity site binding, do not depend on the NsrR [4Fe–4S] cluster and binding is not sensitive to NO, suggesting a role for apo‐NsrR in transcriptional regulation.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Proline 107 is a major determinant in maintaining the structure of the distal pocket and reactivity of the high-spin heme of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H(2)O(2) and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase IMPLICATIONS FOR THE CATALYTIC MECHANISM

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O2-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.