Modes of Accessing Bicarbonate for the Regulation of Membrane Guanylate Cyclase (ROS-GC) in Retinal Rods and Cones.

The membrane guanylate cyclase, ROS-GC, that synthesizes cyclic GMP for use as a second messenger for visual transduction in retinal rods and cones, is stimulated by bicarbonate. Bicarbonate acts directly on ROS-GC1, because it enhanced the enzymatic activity of a purified, recombinant fragment of bovine ROS-GC1 consisting solely of the core catalytic domain. Moreover, recombinant ROS-GC1 proved to be a true sensor of bicarbonate, rather than a sensor for CO2. Access to bicarbonate differed in rods and cones of larval salamander, Ambystoma tigrinum, of unknown sex. In rods, bicarbonate entered at the synapse and diffused to the outer segment, where it was removed by Cl--dependent exchange. In contrast, cones generated bicarbonate internally from endogenous CO2 or from exogenous CO2 that was present in extracellular solutions of bicarbonate. Bicarbonate production from both sources of CO2 was blocked by the carbonic anhydrase inhibitor, acetazolamide. Carbonic anhydrase II expression was verified immunohistochemically in cones but not in rods. In addition, cones acquired bicarbonate at their outer segments as well as at their inner segments. The multiple pathways for access in cones may support greater uptake of bicarbonate than in rods and buffer changes in its intracellular concentration.

Ca2+-Sensor Neurocalcin δ and Hormone ANF Modulate ANF-RGC Activity by Diverse Pathways: Role of the Signaling Helix Domain.

Prototype member of the membrane guanylate cyclase family, ANF-RGC (Atrial Natriuretic Factor Receptor Guanylate Cyclase), is the physiological signal transducer of two most hypotensive hormones ANF and BNP, and of the intracellular free Ca2+. Both the hormonal and the Ca2+-modulated signals operate through a common second messenger, cyclic GMP; yet, their operational modes are divergent. The hormonal pathways originate at the extracellular domain of the guanylate cyclase; and through a cascade of structural changes in its successive domains activate the C-terminal catalytic domain (CCD). In contrast, the Ca2+ signal operating via its sensor, myristoylated neurocalcin δ both originates and is translated directly at the CCD. Through a detailed sequential deletion and expression analyses, the present study examines the role of the signaling helix domain (SHD) in these two transduction pathways. SHD is a conserved 35-amino acid helical region of the guanylate cyclase, composed of five heptads, each meant to tune and transmit the hormonal signals to the CCD for their translation and generation of cyclic GMP. Its structure is homo-dimeric and the molecular docking analyses point out to the possibility of antiparallel arrangement of the helices. Contrary to the hormonal signaling, SHD has no role in regulation of the Ca2+- modulated pathway. The findings establish and define in molecular terms the presence of two distinct non-overlapping transduction modes of ANF-RGC, and for the first time demonstrate how differently they operate, and, yet generate cyclic GMP utilizing common CCD machinery.

Experimental Approaches for Defining the Role of the Ca2+-Modulated ROS-GC System in Retinal Rods of Mouse.

Our ability to see is based on the activity of retinal rod and cone photoreceptors. Rods function when there is very little light, while cones operate at higher light levels. Photon absorption by rhodopsin activates a biochemical cascade that converts photic energy into a change in the membrane potential of the cell by decreasing the levels of a second messenger, cGMP, that control the gating of cation channels. But just as important as the activation of the cascade are the shut-off and recovery processes. The timing of shutoff and recovery ultimately affects sensitivity, temporal resolution and even the capacity for counting single photons. An important part of the recovery is restoration of cGMP through the action of rod outer segment membrane guanylate cyclases (ROS-GCs) and guanylate cyclase-activating proteins (GCAPs). In darkness, ROS-GCs catalyze the conversion of GTP to cGMP at a low rate, due to inhibition of cyclase activity by GCAPs. In the light, GCAP enhances ROS-GC activity. Mutations in the ROS-GC system can cause problems in vision, and even result in blindness due to photoreceptor death. The mouse has emerged as a particularly useful subject to study the role of ROS-GC because the technology for the manipulation of their genetics is advanced, making production of mice with targeted mutations much easier. Here we describe some experimental procedures for studying the retinal rods of wild-type and genetically engineered mice: biochemical assays of ROS-GC activity, immunohistochemistry, and single cell recording.

Membrane Guanylate Cyclase catalytic Subdomain: Structure and Linkage with Calcium Sensors and Bicarbonate.

Membrane guanylate cyclase (MGC) is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD) module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC's CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1) The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2) It exists as a homo-dimer and contains seven conserved catalytic elements (CEs) wedged into seven conserved motifs. (3) It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V836-L857. (4) Site-directed mutagenesis documents that each of the seven CEs governs the cyclase's catalytic activity. (5) In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6) Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD) to exhibit its major (∼92%) catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7) the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the physiological activity of other members of MGC family.

Bicarbonate and Ca(2+) Sensing Modulators Activate Photoreceptor ROS-GC1 Synergistically.

Photoreceptor ROS-GC1, a prototype subfamily member of the membrane guanylate cyclase family, is a central component of phototransduction. It is a single transmembrane-spanning protein, composed of modular blocks. In rods, guanylate cyclase activating proteins (GCAPs) 1 and 2 bind to its juxtamembrane domain (JMD) and the C-terminal extension, respectively, to accelerate cyclic GMP synthesis when Ca(2+) levels are low. In cones, the additional expression of the Ca(2+)-dependent guanylate cyclase activating protein (CD-GCAP) S100B which binds to its C-terminal extension, supports acceleration of cyclic GMP synthesis at high Ca(2+) levels. Independent of Ca(2+), ROS-GC1 activity is also stimulated directly by bicarbonate binding to the core catalytic domain (CCD). Several enticing molecular features of this transduction system are revealed in the present study. In combination, bicarbonate and Ca(2+)-dependent modulators raised maximal ROS-GC activity to levels that exceeded the sum of their individual effects. The F(514)S mutation in ROS-GC1 that causes blindness in type 1 Leber's congenital amaurosis (LCA) severely reduced basal ROS-GC1 activity. GCAP2 and S100B Ca(2+) signaling modes remained functional, while the GCAP1-modulated mode was diminished. Bicarbonate nearly restored basal activity as well as GCAP2- and S100B-stimulated activities of the F(514)S mutant to normal levels but could not resurrect GCAP1 stimulation. We conclude that GCAP1 and GCAP2 forge distinct pathways through domain-specific modules of ROS-GC1 whereas the S100B and GCAP2 pathways may overlap. The synergistic interlinking of bicarbonate to GCAPs- and S100B-modulated pathways intensifies and tunes the dependence of cyclic GMP synthesis on intracellular Ca(2+). Our study challenges the recently proposed GCAP1 and GCAP2 "overlapping" phototransduction model (Peshenko et al., 2015b).

Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca(2+).

Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca(2+) modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, ANF- and B-type natriuretic peptide (BNP). After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation, and anti-proliferation. Very recently another modus operandus for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca(2+). The Ca(2+) sensor neurocalcin d mediates this signaling mechanism. Strikingly, the Ca(2+) and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

The ANF-RGC gene motif (669)WTAPELL(675) is vital for blood pressure regulation: biochemical mechanism.

ANF-RGC is the prototype membrane guanylate cyclase, both the receptor and the signal transducer of the hormones ANF and BNP. After binding them at the extracellular domain, it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates production of the second messenger, cyclic GMP. This, in turn, controls the physiological processes of blood pressure, cardiovascular function, fluid secretion, and others: metabolic syndrome, obesity, and apoptosis. The biochemical mechanism by which this single molecule controls these diverse processes, explicitly blood pressure regulation, is the subject of this study. In line with the concept that the structural modules of ANF-RGC are designed to respond to more than one yet distinctive signals, the study demonstrates the construction of a novel ANF-RGC-In-gene-(669)WTAPELL(675) mouse model. Through this model, the study establishes that (669)WTAPELL(675) is a vital ANF signal transducer motif of the guanylate cyclase. Its striking physiological features linked with their biochemistry are the following. (1) It controls the hormonally dependent cyclic GMP production in the kidney and the adrenal gland. Its deletion causes (2) hypertension and (3) cardiac hypertrophy. (4) These mice show higher levels of the plasma aldosterone. For the first time, a mere seven-amino acid-encoded motif of the mouse gene has been directly linked with the physiological control of blood pressure regulation, a detailed biochemistry of this linkage has been established, and a model for this linkage has been described.

Ca(2+) modulation of ANF-RGC: new signaling paradigm interlocked with blood pressure regulation.

ANF-RGC is the prototype receptor membrane guanylate cyclase that is both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single-transmembrane protein. After binding these hormones at the extracellular domain, ANF-RGC at its intracellular domain signals the activation of the C-terminal catalytic module and accelerates the production of the second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. At present, this is the sole transduction mechanism and the physiological function of ANF-RGC. Through comprehensive studies involving biochemistry, immunohistochemistry, and blood pressure measurements in mice with targeted gene deletions, this study demonstrates a new signaling model of ANF-RGC that also controls blood pressure. In this model, (1) ANF-RGC is not the transducer of ANF and BNP, (2) its extracellular domain is not used for signaling, and (3) the signal flow is not downstream from the extracellular domain to the core catalytic domain. Instead, the signal is the intracellular Ca(2+), which is translated at the site of its reception, at the core catalytic domain of ANF-RGC. A model for this Ca(2+) signal transduction is diagrammed. It captures Ca(2+) through its Ca(2+) sensor myristoylated neurocalcin δ and upregulates ANF-RGC activity with a K(1/2) of 0.5 µM. The neurocalcin δ-modulated domain resides in the (849)DIVGFTALSAESTPMQVV(866) segment of ANF-RGC, which is a part of the core catalytic domain. Thereby, ANF-RGC is primed to receive, transmit, and translate the Ca(2+) signals into the generation of cyclic GMP at a rapid rate. The study defines a new paradigm of membrane guanylate cyclase signaling, which is linked to the physiology of cardiac vasculature regulation and possibly also to fluid secretion.

Differential Ca(2+) sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling.

Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ∼1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.

Antithetical modes of and the Ca(2+) sensors targeting in ANF-RGC and ROS-GC1 membrane guanylate cyclases.

THE MEMBRANE GUANYLATE CYCLASE FAMILY HAS BEEN BRANCHED INTO THREE SUBFAMILIES: natriuretic peptide hormone surface receptors, Ca(2+)-modulated neuronal ROS-GC, and Ca(2+)-modulated odorant surface receptor ONE-GC. The first subfamily is solely modulated by the extracellularly generated hormonal signals; the second, by the intracellularly generated sensory and sensory-linked signals; and the third, by combination of these two. The present study defines a new paradigm and a new mechanism of Ca(2+) signaling. (1) It demonstrates for the first time that ANF-RGC, the prototype member of the surface receptor subfamily, is stimulated by free [Ca(2+)](i). The stimulation occurs via myristoylated form of neurocalcin δ, and both the guanylate cyclase and the calcium sensor neurocalcin δ are present in the glomerulosa region of the adrenal gland. (2) The EF-2, EF-3 and EF-4 hands of GCAP1 sense the progressive increment of [Ca(2+)](i) and with a K(1/2) of 100 nM turn ROS-GC1 "OFF." In total reversal, the same EF hands upon sensing the progressive increment of [Ca(2+)](i) with K(1/2) turn ONE-GC "ON." The findings suggest a universal Ca(2+)-modulated signal transduction theme of the membrane guanylate cyclase family; demonstrate that signaling of ANF-RGC occurs by the peptide hormones and also by [Ca(2+)](i) signals; that for the Ca(2+) signal transduction, ANF-RGC functions as a two-component transduction system consisting of the Ca(2+) sensor neurocalcin δ and the transducer ANF-RGC; and that the neurocalcin δ in this case expands beyond its NCS family. Furthermore, the study shows a novel mechanism of the [Ca(2+)](i) sensor GCAP1 where it acts as an antithetical NCS for the signaling mechanisms of ROS-GC1 and ONE-GC.

S100B serves as a Ca(2+) sensor for ROS-GC1 guanylate cyclase in cones but not in rods of the murine retina.

Rod outer segment membrane guanylate cyclase (ROS-GC1) is a bimodal Ca(2+) signal transduction switch. Lowering [Ca(2+)](i) from 200 to 20 nM progressively turns it "ON" as does raising [Ca(2+)](i) from 500 to 5000 nM. The mode operating at lower [Ca(2+)](i) plays a vital role in phototransduction in both rods and cones. The physiological function of the mode operating at elevated [Ca(2+)](i) is not known. Through comprehensive studies on mice involving gene deletions, biochemistry, immunohistochemistry, electroretinograms and single cell recordings, the present study demonstrates that the Ca(2+)-sensor S100B coexists with and is physiologically linked to ROS-GC1 in cones but not in rods. It up-regulates ROS-GC1 activity with a K(1/2) for Ca(2+) greater than 500 nM and modulates the transmission of neural signals to cone ON-bipolar cells. Furthermore, a possibility is raised that under pathological conditions where [Ca(2+)](i) levels rise to and perhaps even enter the micromolar range, the S100B signaling switch will be turned "ON" causing an explosive production of CNG channel opening and further rise in [Ca(2+)](i) in cone outer segments. The findings define a new cone-specific Ca(2+)-dependent feature of photoreceptors and expand our understanding of the operational principles of phototransduction machinery.

657WTAPELL663 motif of the photoreceptor ROS-GC1: a general phototransduction switch.

This study documents the identity of an intriguing transduction mechanism of the [Ca(2+)](i) signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca(2+) transmitter switch (Ca(2+)TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is (657)WTAPELL(663). The two Ca(2+) signaling GCAP pathways converge in Ca(2+)TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca(2+)-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.

Ca(2+) sensor GCAP1: A constitutive element of the ONE-GC-modulated odorant signal transduction pathway.

In a small subset of the olfactory sensory neurons, the odorant receptor ONE-GC guanylate cyclase is a central transduction component of the cyclic GMP signaling pathway. In a two-step transduction model, the odorant, uroguanylin, binds to the extracellular domain and activates its intracellular domain to generate the odorant second messenger, cyclic GMP. This study via comprehensive technology, including gene deletion, live cell Forster resonance energy transfer (FRET), and surface plasmon resonance (SPR) spectroscopy, documents the identity of a remarkably intriguing operation of a Ca(2+) sensor component of the ONE-GC transduction machinery, GCAP1. In the ciliary membranes, the sites of odorant transduction, GCAP1 is biochemically and physiologically coupled to ONE-GC. Strikingly, this coupling reverses its well- established function in ROS-GC1 signaling, linked with phototransduction. In response to the free Ca(2+) range from nanomolar to semimicromolar, it inhibits ROS-GC1, yet in this range, it incrementally stimulates ONE-GC. These two opposite modes of signaling two SENSORY processes by a single Ca(2+) sensor define a new transduction paradigm of membrane guanylate cyclases. This paradigm is pictorially presented.

Hippocalcin, new Ca(2+) sensor of a ROS-GC subfamily member, ONE-GC, membrane guanylate cyclase transduction system.

Hippocalcin is a member of the neuronal Ca(2+) sensor protein family. Among its many biochemical functions, its established physiological function is that via neuronal apoptosis inhibitory protein it protects the neurons from Ca(2+)-induced cell death. The precise biochemical mechanism/s, through which hippocalcin functions, is not clear. In the present study, a new mechanism by which it functions is defined. The bovine form of hippocalcin (BovHpca) native to the hippocampus has been purified, sequenced, cloned, and studied. The findings show that there is the evolutionary conservation of its structure. It is a Ca(2+)-sensor of a variant form of the ROS-GC subfamily of membrane guanylate cyclases, ONE-GC. It senses physiological increments of Ca(2+) with a K(1/2) of 0.5 microM and stimulates ONE-GC or ONE-GC-like membrane guanylate cyclase. The Hpca-modulated ONE-GC-like transduction system exists in the hippocampal neurons. And hippocalcin-modulated ONE-GC transduction system exists in the olfactory receptor neuroepithelium. The Hpca-gene knock out studies demonstrate that the portion of this is about 30% of the total membrane guanylate cyclase transduction system. The findings establish Hpca as a new Ca(2+) sensor modulator of the ROS-GC membrane guanylate cyclase transduction subfamily. They support the concept on universality of the presence and operation of the ROS-GC transduction system in the sensory and sensory-linked neurons. They validate that the ROS-GC transduction system exists in multiple forms. And they provide an additional mechanism by which ROS-GC subfamily acts as a transducer of the Ca(2+) signals originating in the neurons.

New approaches to understanding double-stranded RNA processing by ribonuclease III purification and assays of homodimeric and heterodimeric forms of RNase III from bacterial extremophiles and mesophiles.

Ribonuclease III (RNase III) is a double-stranded (ds)-RNA-specific endonuclease that plays essential roles in the maturation and decay of coding and noncoding RNAs. Bacterial RNases III are structurally the simplest members of the RNase III family, which includes the eukaryotic orthologs Dicer and Drosha. High-resolution crystal structures of RNase III of the hyperthermophilic bacteria Aquifex aeolicus and Thermotoga maritima are available. A. aeolicus RNase III also has been cocrystallized with dsRNA or specific hairpin substrates. These structures have provided essential structural insight to the mechanism of dsRNA recognition and cleavage. However, comparatively little is known about the catalytic behaviors of A. aeolicus or T. maritima RNases III. This chapter provides protocols for the purification of A. aeolicus and T. maritima RNases III and also describes the preparation of artificial heterodimers of Escherichia coli RNase III, which are providing new insight on the subunit and domain interactions involved in dsRNA recognition and cleavage.

Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III.

Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, muR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of muR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db 'set' is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate.

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis.

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg2+ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg2+ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n (H)) is 2.0 +/- 0.1, indicative of the involvement of two Mg2+ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione--an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites--inhibits E.coli RNase III cleavage of R1.1 RNA. The IC50 for the compound is 14 microM for the Mg2+-supported reaction, and 8 microM for the Mn2+-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.