Interaction of PqqE and PqqD in the pyrroloquinoline quinone (PQQ) biosynthetic pathway links PqqD to the radical SAM superfamily.

pqqD is one of six genes required for PQQ production in Klebsiella pneumoniae. Herein, we demonstrate that PqqD interacts specifically with the radical SAM enzyme PqqE, causing a perturbation in the electronic environment around the [4Fe-4S](+) clusters. This interaction redirects the role for PqqD in PQQ biosynthesis.

Pyrroloquinoline quinone biogenesis: demonstration that PqqE from Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme.

Biogenesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae requires the expression of six genes (pqqA-F). One of these genes (pqqE) encodes a 43 kDa protein (PqqE) that plays a role in the initial steps in PQQ formation [Veletrop, J. S., et al. (1995) J. Bacteriol. 177, 5088-5098]. PqqE contains two highly conserved cysteine motifs at the N- and C-termini, with the N-terminal motif comprised of a CX(3)CX(2)C consensus sequence that is unique to a family of proteins known as radical S-adenosyl-l-methionine (SAM) enzymes [Sofia, H. J., et al. (2001) Nucleic Acids Res. 29, 1097-1106]. PqqE from K. pneumoniae was cloned into Escherichia coli and expressed as the native protein and with an N-terminal His(6) tag. Anaerobic expression and purification of the His(6)-tagged PqqE results in an enzyme with a brownish-red hue indicative of Fe-S cluster formation. Spectroscopic and physical analyses indicate that PqqE contains a mixture of Fe-S clusters, with the predominant form of the enzyme containing two [4Fe-4S] clusters. PqqE isolated anaerobically yields an active enzyme capable of cleaving SAM to methionine and 5'-deoxyadenosine in an uncoupled reaction (k(obs) = 0.011 +/- 0.001 min(-1)). In this reaction, the 5'-deoxyadenosyl radical either abstracts a hydrogen atom from a solvent accessible position in the enzyme or obtains a proton and electron from buffer. The putative PQQ substrate PqqA has not yet been shown to be modified by PqqE, implying that PqqA must be modified before becoming the substrate for PqqE and/or that another protein in the biosynthetic pathway is critical for the initial steps in PQQ biogenesis.

Single- and two-photon properties of a dye-derivatized Roussin's red salt ester (Fe2(mu-RS)2(NO)4) with a large TPA cross section.

The synthesis, characterization, photochemistry, and two-photon photophysical properties of a new dye-derivatized iron sulfur nitrosyl cluster Fe2(mu-RS)2(NO)4 (AFX-RSE, RS = 2-thioethyl ester of N-phenyl-N-(3-(2-ethoxy)phenyl)-7-(benzothiazol-2-yl)-9,9-diethyl-fluoren-2-yl-amine) were investigated. Under continuous photolysis, AFX-RSE decomposes with modest quantum yields (Phi(diss) = (4.9 +/- 0.9) x 10(-3) at lambda(irr) = 436 nm) as measured from the loss of the nitrosyl bands in the IR absorbance spectrum. Nitric oxide (NO) was qualitatively demonstrated to be photochemically produced via single-photon excitation through the use of an NO-specific electrode. Steady-state luminescence measurements have shown that AFX-RSE fluorescence is about 88% quenched relative to the model compound AF-tosyl. This is attributed to a relatively efficient energy transfer from the excited states of the antenna chromophores to the dinuclear metal center, with the subsequent production of NO. In addition, the two-photon absorption (TPA) cross sections (delta) were measured for the AF-chromophores via the two-photon excitation (TPE) photoluminescence technique using a femtosecond excitation source. The TPA cross section of AFX-RSE was found with this technique to be delta = 246 +/- 8 GM (1 GM = 10(-50) cm4 s photon(-1) molecule(-1)).

A two-photon antenna for photochemical delivery of nitric oxide from a water-soluble, dye-derivatized iron nitrosyl complex using NIR light.

The experiments described here demonstrate the use of two-photon excitation (TPE) to sensitize nitric oxide (NO) release from a dye-derivatized iron/sulfur/nitrosyl cluster Fe2(mu-RS)2(NO)4 (Fluor-RSE, RS = 2-thioethyl ester of fluorescein) with near-infrared (NIR) light in the form of femtosecond pulses from a Ti:sapphire laser. TPE at 800 nm leads both to weak fluorescence from the organic chromophore at lambda(max) = 532 nm and to NO labilization from the cluster. Since the emission from the reference compound Fluor-Et (the ethyl ester of fluorescein) under identical conditions (50/50 CH3CN/phosphate buffer (1 mM) at pH 7.4) is considerably more intense, the weaker emission from Fluor-RSE and the NO generation indicate that the fluorescein excited states initially formed by TPE are largely quenched by energy transfer to the cluster core. The two-photon absorption (TPA) cross section of Fluor-RSE at 800 nm was determined to be delta = 63 +/- 7 GM via the TPA photoluminescence technique. This can be compared to the TPA cross section of 36 GM reported for fluorescein dye in pH 11 aqueous solution and of 32 +/- 3 GM for Fluor-Et measured under conditions comparable to those used for Fluor-RSE. Pulse intensity dependence studies showed that the quantity of NO released from the latter as the result of NIR photoexcitation follows a quadratic relationship to excitation intensity, consistent with the expectation for a TPE process. These studies demonstrate the potential utility of a two-photon antenna for sensitization of the photochemical release of an active agent (in this case, NO) from a photoactive pro-drug.

Toward development of water soluble dye derivatized nitrosyl compounds for photochemical delivery of NO.

This report describes the synthesis, spectroscopy, and photochemistry of a new fluorescein-derivatized iron sulfur nitrosyl compound, the Roussin's red salt ester bis-((mu-S,mu-S')-fluorescein-2-thioethyl-ester)-tetranitrosyldiiron (Fluor-RSE). Under continuous photolysis Fluor-RSE decomposes with moderate quantum yields (0.0036 +/- 0.0005 at lambda(irr) = 436 nm) with the corresponding release of most of the NO carried by the Fe2S2NO4 cluster. Large changes in the optical absorptivity occur upon photolysis of the Fluor-RSE, and these changes have been attributed to the different protic forms available to the fluorescein chromophore as it is separated from the cluster. Steady-state luminescence experiments have shown that the fluorescence of Fluor-RSE is about 85% quenched relative to the model compound ethyl fluorescein (Fluor-Et). Thus, it is clear that excitation of the fluorescein chromophore antennae is followed by energy transfer to the Fe/S/NO cluster at a rate at least comparable to fluorescence. However, the effect of the iron-sulfur core on the fluorescent lifetimes from fluorescein chromophore is much smaller. A single-exponential decay (tau = 3.3 ns) was seen for Fluor-RSE that is only modestly shorter than that for Fluor-Et (tau = 4.5 ns), and this is the effect of the smaller radiative rate constant (k(r)) for the former. These systems further demonstrate that attachment of a pendant dye chromophore as an antenna significantly improves the effective rate for photochemical NO generation from the Roussin's red salt esters at longer excitation wavelengths.

Probing shapes of bichromophoric metal-organic complexes using ion mobility mass spectrometry.

Ion mobility mass spectrometry (IM-MS) was used to probe the structures of several metal complexes carrying pendant chromophores. The three complexes investigated were the copper(II) complex Cu(DAC)2+ (DAC = 1,8-bis(9-methylanthracyl)cyclam, cyclam = 1,4,8,11-tetraazacyclotetradecane), the N-nitrosylated ligand DAC-NO, and the Roussin's red salt ester (mu-S,mu-S')-protoporphyrin-IX-bis(2-thioethyl ester)tetranitrosyldiiron (PPIX-RSE). From the IM-MS data coupled with theoretical calculations, it was found that [Cu(II)(DAC - H)]+ exists as a single conformer, with one anthracenyl group above the cyclam and the other below, similar to the crystal structure of Cu(II)(DAC)2+. The metal-free N-nitrosylated ligand (DAC-NO + H)+ has two conformations: one family of structures has one anthracenyl group above the cyclam and one below, while the other has both anthracenyl groups on the same side of the cyclam. These observations are consistent with 1H NMR data for the neutral DAC-NO complex that indicate the presence of two geometric isomers in solution. The third species, PPIX-RSE, has a porphyrin chromophore covalently linked to an Fe2S2(NO)4 cluster for use as a precursor for the photochemical delivery of nitric oxide in single- and two-photon excitation processes. Ion mobility indicates the presence of two (PPIX-RSE + H)+ conformations, consistent with the previous interpretation of the bimodal fluorescence lifetime decay seen for PPIX-RSE. DFT structures, in good agreement with the IM-MS cross sections, indicate two "bent" conformations with the planes of the porphyrin and Fe2S2 rings at different angles with respect to each other.

Photochemical production of nitric oxide via two-photon excitation with NIR light.

Herein we demonstrate the successful photochemical generation of nitric oxide via two-photon excitation (TPE) from the supramolecular complex PPIX-RSE ({mu-S,mu-S'-protoporphyrin-IX-bis(2-thioethyl)diester]tetranitrosyl-diiron). The TPE fluorescence spectra indicate efficient energy transfer from the PPIX antennae to the iron sulfur nitrosyl cluster. Further evidence of NO release is demonstrated using a nitric oxide specific electrode and ESI+ MS.

Synthesis and photochemical properties of a novel iron-sulfur-nitrosyl cluster derivatized with the pendant chromophore protoporphyrin IX.

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

Photochemical investigation of Roussin's red salt esters: Fe2(mu-SR)2(NO)4.

The photochemistry of various Roussin's red ester compounds of the general formula Fe(2)(SR)(2)(NO)(4), where R = CH(3), CH(2)CH(3), CH(2)C(6)H(5), CH(2)CH(2)OH, and CH(2)CH(2)SO(3)(-), were investigated. Continuous photolyses of these ester compounds in aerated solutions led to the release of NO with moderate quantum yields for the photodecomposition of the ester (Phi(RSE) = 0.02-0.13). Electrochemical studies using an NO electrode demonstrated that 4 mol of NO are generated for each mole of ester undergoing photodecomposition. Nanosecond flash photolysis studies of Fe(2)(SR)(2)(NO)(4) (where R = CH(2)CH(2)OH and CH(2)CH(2)SO(3)(-)) indicate that the initial photoreaction is the reversible dissociation of NO. In the absence of oxygen, the presumed intermediate, Fe(2)(SR)(2)(NO)(3), undergoes second-order reaction with NO to regenerate the parent cluster with a rate constant of k(NO) = 1.1 x 10(9) M(-1) s(-1) for R = CH(2)CH(2)OH. Under aerated conditions the intermediate reacts with oxygen to give permanent photochemistry.