Extensive minimization and dynamics computational studies of the hammerhead structural domain of the virusoid of lucerne transient streak virus have been carried out. The following observations at the self-cleavage position are derived from the resulting three-dimensional structure: (1) the cytosine base is at the surface and does not interact with another base (it is free to move), and (it) the ribose-phosphate backbone is forced to take an abrupt turn since it bridges stems I and III, and this turn points the pro-S and pro-R oxygens of the phosphate to the inward side of the hammerhead. These structural features are independent of the hammerhead being open or closed and allow an unencumbered 3'-to 2'-endo conformational change of the ribose with the resultant creation of an unusual stereochemistry that allows a direct in-line self-cleavage reaction. In the closed hammerhead structure, interactions of stems I and II create a vacancy into which the catalytic hydrated Mg(II) may be docked on labile phosphate. This opening is not present if stems I and II are shortened.Certain single-stranded, circular RNAs undergo a sitespecific self-cleavage reaction during replication. The selfcleavage reactions occur in a common structural domain known as a hammerhead (1, 2). The hammerhead structure is sufficient for self-cleavage in avocado sunblotch viroid and in virusoid of lucerne transient streak virus (structure A) and To understand the mechanism of self-cleavage the threedimensional structure of the hammerhead (as A) must be known. The use of x-ray and NMR techniques is plausible with stable hammerhead structures; however, these RNA molecules are in a noncleaving conformation (7). For those hammerhead molecules that do cleave, the cleavage rate is too great for the use of spectroscopic methods. In any event, with the exception of tRNAPhe (8) and tRNAASP (9), crystallographic methods have not been applicable for the determination of RNA structure. Chemical modifications in the vicinity of the cleavage site may allow NMR structural determinations, but the catalytic structural tuning of the self-cleavage site may be lost. Herein we present a computational approach to the wild-type hammerhead structure A, using the molecular mechanics programs of CHARMM (10). Our goal is to determine whether the three-dimensional structure obtained by this means will allow the formulation of a rational mechanism of self-cleavage that might be used as a working hypothesis. We assure the reader that we are aware of the limitations of this computational approach to mechanism.Nilsson and Karplus ( most likely is sufficient in newt satellite RNA (3). In the self-cleaving hammerhead structure there are 13 conserved nucleotides (boxed) and cleavage is always at the same phosphodiester linkage (arrow). Self-cleavage involves nucleophilic displacement by a 2'-hydroxyl upon the adjacent 3'-phosphate to generate a 2',3'-cyclic phosphate ester with the release of a 5'-hydroxyl group. In this respect the self-cleavage reaction resembles that of...

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It has been proposed in the literature that the chemiluminescence of the flavoenzyme of bacterial luciferase is caused by a chemically initiated electron-exchange luminescence mechanism which provides an excited 4a-hydroxy-4a,5-dihydroflavin ([4a-FlHOH]*) as product of 1e- reduction of the radical 4a-FlHOH+.. Electrochemical/photon counting experiments were performed to assess the feasibility of this proposal. Potentials for step-wise oxidation of N(5)-ethyl-4a-hydroxy-4a,5-dihydroluminflavin (4a-FlEtOH) have been determined in dry N,N-dimethylformamide (DMF). Photon counting was carried out during the 1e- reduction of 4a-FlEtOH+.in both DMF and acetonitrile by use of an apparatus consisting of a photocell mounted below a Pt ring-disk electrode. By use of the ring-disk electrode a steady state concentration of [4a-FlEtOH]* could be maintained by continuous 1e- oxidation of 4a-FlEtOH----4a-FlEtOH+.and 1e- reduction of 4a-FlEtOH+.----4a-FlEtOH. A maximum of 14% collection (theoretical maximum is 18%) of FlEtOH.+ at the ring electrode was obtained below 5000 rotations per minute. Calibration of the apparatus using 9,10-diphenylanthracene allowed approximation of the quantum yield for 1e- reductive capture of 4a-FlEtOH+.as 10(-6) to 10(-4) in DMF and 10(-7) to 10(-5) in acetonitrile. No fluorescence for 4a-FlEtOH in DMF could be observed; if fluorescent, the efficiency of 4a-FlEtOH can be no greater than approximately 3 x 10(-5). No electrogenerated chemiluminescence is observed on the electrochemical recycling of FlEt+----FlEt2+ and FlEt2+----FlEt+.

The nature and rate of reduction of H2+ toHgo by 1,5-dihydro-3,(3-sulfopropyl)lumiflavin (FIlH2) in buffered aqueous solutions (pH 4.7) is dependent on the ligation of Hg2e. In the presence of NN-bis(2-hydroxyethyl)glycine or when ligated to ethylenediaminetetraacetic acid, the reduction is first order in Hg2+ and FIH2. The apparent second-order rate constant with N,N-bis(2-hydroxyethyl)glycine (2.2 x 10' M-l s'l) is much greater than that in the presence of ligating ethylenediaminetetraacetic acid (1.5 x 102 M'-s-'). 10 (3, 4, 6). How an enzymatic protonolysis shows maximum activity at low molar hydrogen ion concentration ([H+]) is an intriguing question. Organomercurial Iyase activity requires the presence of a -2-fold excess of thiol over RHgX substrate. We have shown that bis-thiol-ligation provides a 103-fold rate enhancement in the protonolysis of an arylmercurial in aqueous solution (7). The protonolysis reaction was first order in both buffer and the bis-thiol-ligated organomercurial. The apparent second-order rate constants exhibited bell-shaped pH vs. rate-constant profiles because of a change of the rate-determining step on increase in pH. A kinetically competent mechanism involves coordination ofbuffer base to the bis-ligated organomercurial followed by specific acid cleavage of the carbon-mercury bond (Eq. 1). The pH optimum is determined by the pKa of the conjugate acid of the buffer base B; and the protonolysis reaction rate is amplified by bis-thiol-ligation of the mercuric moiety of the substrate. A similar mechanism was proposed for organomercurial Iyase (7). by the resultant EH2-NADPH is followed by a second 2e-reduction to give EH4-NADP+ (12, 13). The 1,5-dihydroflavin (FADH2) moiety of EH4-NADP', in a nonrate-limiting step, reduces Hg2+ (13,14). Mercury(II) reductase enzymes differ from the glutathione reductase family of enzymes in that the latter contain a second conserved cysteine pair (15). The proposal has been put forth that ligation of Hg2' by mercury(II) reductase involves both cysteine pairs so that complexation of Hg2' by a third and/or fourth thiol ligand substantially changes the nature of the mercury(II)-sulfur bonding by raising the reduction potential of complexed Hg2' and making its reduction more facile (16). Abbreviations: FIH2, 1,5-dihydro-3(3-sulfopropyl)lumiflavin; Fl0", 3(3-sulfopropyl)lumiflavin; DMPS, 2,3-dimercapto-1-propanesulfonate; Bicine, N,N-bis(2-hydroxyethyl)glycine. 3041The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.