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.

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 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 consensus sequence of CX 3 CX 2 C that is unique to a family of proteins known as radical Sadenosyl-L-methionine (SAM) enzymes (Sofia et al. (2001) Nucleic Acids Res. 29, 1097-1106. PqqE from K. pneumoniae was cloned into E. coli and expressed as the native protein and with an Nterminal His 6 -tag. Anaerobic expression and purification of the His 6 -tag 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 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 either 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. KeywordsQuinones; cofactor; biogenesis; S-adenosyl-methionine; pyrroloquinoline quinone; radicals; ironsulfur clusters † This work was supported by research grants from the National Institutes of Health (GM39296 to JPK, GM073789 to DB and F32GM080795 to SRW) and Howard Hughes Medical Institute (to DK). *To whom correspondence should be sent: tel: 510-642-7460; fax: 510-643-6232; klinman@berkeley.edu. # Present address: Decode Genetics, Sturlagata 8, IS-101 Reykjavik, Iceland. Δ Present address: Department of Applied Chemistry, National Chiao Tung University, 1001 Hsueh Road, Hsinchu, Taiwan, Republic of China.Supporting Information Available: Methods for the aerobic and anaerobic growth and induction of E. coli BL21(DE3) cells harboring the pET24b-pqqE plasmid, aerobic purification of PqqE, anaerobic reconstitution of PqqE with iron and sulfide ions, synthesis and purification of SAM, and a detailed description of the high resolution mass spectrometry experiments. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 October 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe broad spectrum of chemical reactions catalyzed by enzymes frequently requires functional groups that are unavailable from the side chains of the 20 naturally occurring amino acids. In particula...

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.

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.