pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

Mutation of Arginine 134 to Lysine Alters the pKas of Key Groups Involved in Proton Pumping by Bacteriorhodopsin

Abstract— Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK_as of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counter-ion, D85, and of the proton release group. We calculate, in R134K, that the pK_a of D85 is 4.6 in the unphotolyzed state, while the pK_a of the proton release group is 8.0 in the unphotolyzed state but drops to ∽5.8 in the M intermediate. The same value for the pK_a of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release.
The altered values of these pK_as relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

Fiber optic pH sensing with long wavelength excitable Schiff bases in the pH range of 7.0-12.0

Most of the fluorescent pH probes work near neutral or acidic regions of the pH scale. In this work, two different fluorescent Schiff bases, chloro phenyl imino propenyl aniline (CPIPA) and nitro phenyl imino propenyl aniline (NPIPA), have been investigated for pH sensing in the alkaline region. Absorption and emission based spectral data, quantum yield, fluorescence lifetime, photostability and acidity constant (pK(a)) of the Schiff bases were determined in conventional solvents and in PVC. The long wavelength excitable immobilized Schiff bases CPIPA (lambda(ex)=556 nm) and NPIPA (lambda(ex)=570 nm) exhibited absorption and emission based optical response to proton in the pH range of 8.0-12.0 and 7.0-12.0, respectively. Response of the CPIPA was fully reversible within the dynamic working range. The response times were between 3-13 min. A relative signal change of 95% and 96% have been achieved for sensor dyes of CPIPA and NPIPA, respectively. The CPIPA displayed better fluorescence quantum yield (varphi(F)=3.7 x 10(-1)) and higher matrix compatibility compared to NPIPA (varphi(F)=1.6 x 10(-1)) in immobilized PVC. The CPIPA and NPIPA exhibited a slight cross sensitivity to the ions of Hg(+) and Fe(3+), respectively.     

Coloured complexes of all-trans-retinal with benzocaine and other local anaesthetics

Coloured Schiff base complexes with lambda max values of 500 nm or above were formed between the visual pigment all-trans-retinal and the local anesthetics procaine (lambda max = 533 nm), benzocaine (4-amino-benzoic acid ethyl ester, lambda max = 535 nm), 3-amino-benzoic acid ethyl ester (lambda max = 500 nm) and 2-amino-benzoic acid ethyl ester (lambda max = 509 nm) in methanol acidified with HCl. The anaesthetics lidocaine and urethane failed to form coloured compounds with lambda max values greater than 500 nm under the same conditions. The relevance of these observations to the effect of anaesthetics on the visual pigments is discussed.     

THE TWO CONSECUTIVE M SUBSTATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN ARE AFFECTED SPECIFICALLY BY THE D85N AND D96N RESIDUE REPLACEMENTS

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.     

A PROTON RELEASE SITE ON THE C-TERMINAL SIDE OF BACTERIORHODOPSIN

We have studied the pH dependence of the light-induced proton release and uptake by bacteriorhodopsin. The quantum efficiency of proton release in cell envelopes and proton uptake in phospholipid vesicles is high in the low pH range and begins to decline between pH 6 and 7 in cell envelopes and between pH 7–8 in phospholipid vesicles. In the cell envelope vesicles the proton release increases again above pH 8–8.5; in phospholipid vesicles a proton release is observed before proton uptake at pHs greater than 9. We suggest that the light-induced proton release observed at high pHs are due to protons released and rebound on the carboxyl terminal side of bacteriorhodopsin.     

Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone-carbocyanine dye system

Synthetic red and near-infrared absorbing dyes may be used as probe molecules in a large number of applications. Dyes exhibiting spectral changes with hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxima in the long wavelength region of the visible spectral region are specially valuable because of decreased interference and semiconductor laser applications. In this paper we have evaluated an aminodienone dyes 1 which demostrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye is in the reduced interference region. The absorption maximum is at 535 nm in neutral or alkaline solutions in methanol. The absorption spectra undergo a strong bathochromic shift in the presence of acids (lambda(max) = 709 nm) with a concomitant change in the fluorescence spectra. This pH sensitive dye was found to be specially especially useful for organic solvents. The analytical utility of this and similar near-infrared absorbing dyes is discussed.     

An iridium(III) complex that exhibits dual mechanism nonlinear absorption

The photophysical properties of the complex (L)Ir(ppy)(2)(+), where ppy = 2-phenylpyridine and L = 4,4'-(2,2'-bipyridine-5,5'-diylbis(ethyne-2,1-diyl))bis(N,N-dihexylaniline), have been investigated under one- and two-photon excitation conditions. In THF solution, the complex exhibits broad ground-state absorption with lambda(max) approximately 500 nm and weak photoluminescence with lambda(max) approximately 730 nm. Excitation of (L)Ir(ppy)(2)(+) at 355 nm produces a long-lived excited state (tau approximately 1 mus) that features a strong excited-state absorption in the near-infrared (lambda(max) approximately 875 nm, Deltaepsilon approximately 6.1 x 10(4) M(-1) cm(-1)). Photoluminescence and transient absorption studies of (L)Ir(ppy)(2)(+) carried out using 5 ns, 1064 nm pulsed excitation demonstrate that the same long-lived and strongly absorbing excited state can be efficiently produced by two-photon absorption. Solutions of the complex in THF display nonlinear absorption of 5 ns, 1064 nm pulses in a process that is believed to involve a combination of two-photon absorption and reverse saturable absorption.     

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.     

REGENERATION OF BLUE AND PURPLE MEMBRANES FROM DEIONIZED BLEACHED MEMBRANES OF Halobacterium halobium

Abstract— Bleached purple membrane normally binds Ca²⁺ and Mg²⁺, which can be removed by the divalent cation chelator ethylenediaminetetraacetic acid (EDTA). Regeneration of pigments from EDTA-treated bleached membrane (apomembrane) and retinal leads to the formation of blue membrane at pH 4.8, and purple membrane at neutral pH. The pigments take much longer to regenerate than with un-deionized apoprotein. Adding back cations to the deionized apomembrane only partially speeds up the regeneration process. Like native purple membrane, the regenerated purple membrane also undergoes a photocycle and shows a light-induced proton release and uptake, although with much slower kinetics than the native species. Thus, cations control the kinetics of pigment regeneration, and also some aspects of the pigment's conformation which controls the photocycle kinetics. The removal and replacement of the cations is not completely reversible, suggesting the cations are not merely bound in the double layer.     

Time-resolved FTIR spectroscopy of the photointermediates involved in fast transient H+ release by proteorhodopsin

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.     

Photochemical Properties of the Red‐shifted Channelrhodopsin Chrimson

Color‐tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light‐induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground‐state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.     

Sensitive circular dichroism marker for the chromophore environment of photoactive yellow protein: assignment of the 307 and 318 nm bands to the n --> pi* transition of the carbonyl

The absorption and CD spectra of wild-type PYP, apo-PYP, and the mutants, E46Q and M100A, were measured between 250 and 550 nm. At neutral pH, the two very weak absorption bands of wild-type PYP at 307 and 318 nm (epsilon(max) = 600 +/- 100 M(-1) cm(-1) at 318 nm) are associated with quite strong positive CD bands (Deltaepsilon(max) approximately 6.8 M(-1) cm(-1)). Both sets of bands are absent in the apoprotein. On the basis of this evidence, we assign these optical signals to the n --> pi* transition of the oxygen of the carbonyl group of the 4-hydroxycinnamic acid chromophore, which is expected to be electric dipole forbidden but magnetic dipole allowed. The progression of narrow bands at 307 and 318 nm with a shoulder in the CD around 329 nm is due to vibrational fine structure with a frequency of about 1050 +/- 50 cm(-1). This is the carbonyl stretch frequency in the electronically excited state and is well-known from the vibrational structure in the CD spectra of carbonyl compounds. The positive sign of the CD in the near UV is in accordance with the octant rule and the high-resolution X-ray structure, if we assume that the NH group of cysteine 69 to which the carbonyl is hydrogen bonded is the principle perturbant. Similar absorption and CD spectra were observed in the range of 300-340 nm for the mutants E46Q and M100A at neutral pH. Protonation of the trans chromophore by lowering the pH in the dark (without photoisomerization) broadens the 307 and 318 nm CD bands in the mutant E46Q but does not significantly affect their positions or alter their sign. For the long-lived I(2) photointermediate of the mutant M100A with protonated cis chromophore, we observed that the sign of the rotational strength in the 310-320 nm range is negative (i.e., opposite to that in the dark state with trans chromophore). This suggests that the light-induced isomerization of the chromophore, which leads to breaking of the hydrogen bond with the backbone amide of C69, brings the carbonyl into a new protein environment with different asymmetry than in the unbleached protein. The observed change in sign is mainly due to this effect, but a change in chromophore twist may also contribute. Thus, the 318 nm CD signal is a sensitive marker for the environment of the chromophore carbonyl, which samples various environments and configurations during the photocycle.     

Reversible proton transfer dynamics in bacteriorhodopsin

In a previous study of ab initio dynamics, the proton transfer in bacteriorhodopsin from protonated asp96 in the cytoplasmic region toward the deprotonated Schiff base was investigated. A quantum mechanics/molecular mechanics model was constructed from the X-ray structure of bacteriorhodopsin E204Q mutant. In this model, asp96, asp85, and thr89 as well as most of the retinal chromophore and the Schiff base link of lys216 were treated quantum mechanically while the rest of the atoms were treated molecular mechanically. A channel was found in the X-ray structure allowing a water chain to form between the asp96 and Schiff base. In the present study, a chain of four waters from asp96 to the Schiff base N coupled with one branching water supports proton transfer as a concerted event in about 3.5 ps. With both a neutral asp85 and a branched water, the dynamics is now found to be more complicated than observed in the initial study for the transition from the photocycle late M state to the N state. Proton transfer is also observed from the Schiff base back to asp96 demonstrating that there is no effective barrier to proton transfer larger than kT in a strong H-bonded network. The binding of the branched water to the four water chains can dynamically hinder the proton transfer.     

An experimental and theoretical investigation of the photophysics of 1-hydroxy-2-naphthoic acid

Photophysical and photochemical properties of 1-hydroxy-2-naphthoic acid (1,2-HNA) have been investigated experimentally by steady state and time domain fluorescence measurements and theoretically by Hartree-Fock (HF), configuration interaction at the single excitation (CIS) level, density functional theoretic (DFT), and semiempirical (AM1) methods. 1,2-HNA exhibits normal fluorescence that depends on its concentration, nature of the solvent, pH, temperature, and wavelength of excitation. It seems to form different emitting species in different media, akin to 3-hydroxy-2-naphthoic acid (3,2-HNA). The large Stokes shifted emission observed at pH 13 is attributed to species undergoing excited-state intramolecular proton transfer. Nonradiative transition seems to increase on protonation and decrease on deprotonation. AM1(PECI=8) calculations predict the absorption maximum (lambda(max) = 335.9 nm) in reasonable agreement with experiment (lambda(max) = 352 nm) for the neutral 1,2-HNA. They also predict a red shift in absorption on protonation and a blue shift on deprotonation as observed experimentally. CIS calculations tend to overestimate the energy gap and hence underestimate the absorption maxima between the ground and the excited electronic states of 1,2-HNA and its protonated and deprotonated forms. However, they do predict correctly that the excited state intramolecular proton transfer is likely to occur in the deprotonated form of 1,2-HNA and not in the neutral and the protonated forms. A single minimum is found in the potential energy profile for the ground state as well as the first excited state of 1,2-HNA and its protonated species. In contrast, a double minimum with a nominal barrier in between is predicted for the ground state and also the first three excited states of the deprotonated species. The keto form of the deprotonated species is found to be slightly less stable than the enol form in all the states investigated.     

Electric-field effects in dry films of D85N and D85,96N mutant bacteriorhodopsin

In the D85N mutant of the protein bacteriorhodopsin (BR), the Schiff base, by which the retinal chromophore is bound to the protein, exhibits an abnormally low proton affinity (pKa approximately 8.9). Recent experiments on thin films of this protein have shown that this causes the protonation state of the Schiff base, and thus the visible absorption spectrum, to be sensitive to external electric fields. In this paper, we explore the dependence of this effect on parameters such as pH, humidity, and film thickness. The results of these experiments point to the importance of water molecules bound in the acceptor part of the proton channel as sources and donors in field-induced proton-transfer reactions. We describe additional results obtained with the D85,96N mutant, which also exhibits a low Schiff-base pK. The similar behavior of the two mutants under applied electric fields at high pH implies that the residue Asp-96 plays no role in field-induced Schiff-base protonation.     

Synthesis and characterization of model compounds of the lysine tyrosyl quinone cofactor of lysyl oxidase

4-n-Butylamino-5-ethyl-1,2-benzoquinone (1(ox)) has been synthesized as a model compound for the LTQ (lysine tyrosyl quinone) cofactor of lysyl oxidase (LOX). At pH 7, 1(ox) has a lambda(max) at 504 nm and exists as a neutral o-quinone in contrast to a TPQ (2,4,5-trihydroxyphenylalanine quinone) model compound, 4, which is a resonance-stabilized monoanion. Despite these structural differences 1(ox) and 4 have the same redox potential (ca. -180 mV vs SCE). The structure of the phenylhydrazine adduct of 1(ox) (2) is reported, and 2D NMR spectroscopy has been used to show that the position of nucleophilic addition is at C(1). UV-vis spectroscopic pH titration of phenylhydrazine adducts of 1(ox) and 4, 2, and 11, respectively, reveals a similar red shift in lambda(max) at alkaline pH with the same pK(a) (approximately 11.8). In contrast, the red shift in lambda(max) at acidic pH conditions yields different pK(a) values (2.12 for 2 vs -0.28 for 11), providing a means to distinguish LTQ from TPQ. Reactions between in situ generated 4-ethyl-1,2-benzoquinone and primary amines give a mixture of products, indicating that the protein environment must play an essential role in LTQ biogenesis by directing the nucleophilic addition of the epsilon-amino group of a lysine residue to the C(4) position of a putative dopaquinone intermediate. Characterization of a 1,6-adduct between an o-quinone and butylamine (3-n-butylamino-5-ethyl-1,2-benzoquinone, 13) confirms the assignment of LTQ as a 1,4-addition product.     

Photolabile ruthenium nitrosyls with planar dicarboxamide tetradentate N(4) ligands: effects of in-plane and axial ligand strength on NO release

Four ruthenium nitrosyls, namely [(bpb)Ru(NO)(Cl)] (1), [(Me(2)bpb)Ru(NO)(Cl)] (2), [(Me(2)bpb)Ru(NO)(py)](BF(4)) (3), and [(Me(2)bqb)Ru(NO)(Cl)] (4) (H(2)bpb = 1,2-bis(pyridine-2-carboxamido)benzene, H(2)Me(2)bpb = 1,2-bis(pyridine-2-carboxamido)-4,5-dimethylbenzene, H(2)Me(2)bqb = 1,2-bis(quinaldine-2-carboxamido)-4,5-dimethylbenzene; H is the dissociable amide proton), have been synthesized and characterized by spectroscopy and X-ray diffraction analysis. All four complexes exhibit nu(NO) in the range 1830-1870 cm(-)(1) indicating the [Ru-NO](6) configuration. Clean (1)H NMR spectra in CD(3)CN (or (CD(3))(2)SO) confirm the S = 0 ground state for all four complexes. Although the complexes are thermally stable, they release NO upon illumination. Rapid NO dissociation occurs when solutions of 1-3 in acetonitrile (MeCN) or DMF are exposed to low-intensity (7 mW) UV light (lambda(max) = 302 nm). Electron paramagnetic resonance (EPR) spectra of the photolyzed solutions display anisotropic signals at g approximately 2.00 that confirm the formation of solvated low-spin Ru(III) species upon NO release. The ligand trans to bound NO namely, anionic Cl(-) and neutral pyridine, has significant effect on the electronic and NO releasing properties of these complexes. Change in the in-plane ligand strength also has effects on the rate of NO release. The absorption maximum (lambda(max)) of 4 is significantly red shifted (455 nm in DMF) compared to the lambda(max) values of 1-3 (380-395 nm in DMF) due to the extension of conjugation on the in-plane ligand frame. As a consequence, 4 is also sensitive to visible light and release NO (albeit at a slower rate) upon illumination to low-intensity visible light (lambda > 465 nm). Collectively, the photosensitivity of the present series of ruthenium nitrosyls demonstrates that the extent of NO release and their wavelength dependence can be modulated by changes of either the in-plane or the axial ligand (trans to bound NO) field strength.