On the photochemical behavior of the [Ru(NH3)4(NO)nicotinamide]3+ cation and the relative stability of light-induced metastable isonitrosyl isomers of Ru complexes.

Low-temperature IR experiments on crystalline samples of trans-[Ru(NH3)4(NO) nicotinamide]3+ salts show a light-induced absorption band typical for MS1 NO linkage isomers upon exposure to 300-500 nm light from a Xe source. The formation of a metastable species is confirmed by DSC measurement on a sample irradiated at low temperature with 457 nm light from an Ar+ laser. The light-induced species decays between 250 and 260 K according to both IR and DSC results. This decay temperature (Td) is somewhat below that observed for other high-Td linkage isomers, even though the NO-stretching frequency of the of [Ru(NH3)4(NO) nicotinamide]3+ ion is above that of the other isomers, demonstrating a lack of precise correlation between the two physical properties. The 90 K crystal structure of trans-[Ru(NH3)4(NO)nicotinamide](SiF6)(NO3).H2O is reported. The geometry from theoretical DFT calculations of the ground-state structure agrees well with the experimental results, except for the orientation of the CONH2 substituent in the pyridine ring, which is rotated by 180 degrees in the crystal due to packing effects. The MS1 and MS2 linkage isomers are found to correspond to local minima on the ground-state potential energy surface, and their geometries and energies are reported.

Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. NiFe(CN)2CO, Biology's way to activate H2.

Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with Mössbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M. , Duin, E. C., Albracht, S. P. J., and Münck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.

Similarities in the architecture of the active sites of Ni-hydrogenases and Fe-hydrogenases detected by means of infrared spectroscopy.

Three groups that absorb in the 2100-1800-cm-1 infrared spectral region have recently been detected in Ni-hydrogenase from Chromatium vinosum [Bagley, K.A., Duin, E.C., Roseboom, W., Albracht, S. P.J. & Woodruff, W.H. (1995) Biochemistry 34, 5527-5535]. To assess the significance and generality of this observation, we have carried out an infrared-spectroscopic study of eight hydrogenases of three different types (nickel, iron and metal-free) and of 11 other iron-sulfur and/or nickel proteins. Infrared bands in the 2100-1800-cm-1 spectral region were found in spectra of all Ni-hydrogenases and Fe-hydrogenases and were absent from spectra of any of the other proteins, including a metal-free hydrogenase. The positions of these bands are dependent on the redox state of the hydrogenase. The three groups in Ni-hydrogenases that are detected by infrared spectroscopy are assigned to the three unidentified small non-protein ligands that coordinate iron in the dinuclear Ni/Fe active site as observed in the X-ray structure of the enzyme from Desulfovibrio gigas [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E.C., Frey, M. & Fontecilla-Camps, J.C. (1995) Nature 373, 580-587]. It is concluded that these groups occur exclusively in metal-containing H2-activating enzymes. It is proposed that the active sites of Ni-hydrogenases and of Fe-hydrogenases have a similar architecture, that is required for the activation of molecular hydrogen.

Infrared-detectable groups sense changes in charge density on the nickel center in hydrogenase from Chromatium vinosum.

Fourier transform infrared studies of nickel hydrogenase from Chromatium vinosum reveal the presence of a set of three absorption bands in the 2100-1900 cm-1 spectral region. These bands, which do not arise from carbon monoxide, have line widths and intensities rivaling those of a band arising from the carbon monoxide stretching frequency (v(CO)) in the Ni(II).CO species of this enzyme [Bagley, K. A., Van Garderen, C. J., Chen, M., Duin, E. C., Albracht, S. P. J., & Woodruff, W. H. (1994) Biochemistry 33, 9229-9236]. The positions of each of these three infrared absorption bands respond in a consistent way to changes in the formal redox state of the nickel center and to the photodissociation of hydrogen bound to the nickel. Up to eight different states of the nickel center have been produced, depending on the redox state and/or the activity state of the enzyme and the presence of carbon monoxide. In seven of these states, the three IR absorption bands in the set have unique frequency positions. It is concluded that the set is due to intrinsic, non-protein groups in the enzyme, whose identities are presently unknown, and that these groups are situated very close to the nickel center and sense the charge density at the Ni site.

Infrared studies on the interaction of carbon monoxide with divalent nickel in hydrogenase from Chromatium vinosum.

Infrared spectra of a carbon monoxy-bound form of the EPR silent Ni(II) species of hydrogenase isolated from Chromatium vinosum are presented. These spectra show a band at 2060 cm-1 due to v(CO) for a metal-CO complex. This absorbance shifts to 2017 cm-1 upon exposure of the enzyme to 13CO. This band is attributed to v(CO) from a Ni(II)-CO species. It is shown that the CO on this species is photolabile at cryogenic temperatures but rebinds to form the original carbon monoxy species at temperatures above 200 K. In addition to the v(CO) band, infrared lines are detected at 2082, 2069, and 1929 cm-1, which shift slightly higher in frequency upon photolysis of the CO from the Ni. These infrared bands do not arise from CO itself on the basis of the fact that the frequency of these bands is unaffected by exposure of the enzyme to 13CO. Experiments in D2O show that these bands do not arise from an exchangeable hydrogen species. It is concluded that these non-CO bands arise from species near or coordinated to the Ni active site. The possible nature of these bands is discussed.

Nature and functional implications of the cytochrome a3 transients after photodissociation of CO-cytochrome oxidase.

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.

A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center.

A comparison is made between the PQA----P+QA- and PQAQB----P+QAQB-transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light-induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm-1 which is present in the P+QA-minus-PQA spectrum and not in the P+QAQB(-)-minus-PQAQB spectrum. In contrast, this signal is present in both P+QA(-)-minus-PQA- and P+QAQB(-)-minus-PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C = O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.

A comparative study of the infrared difference spectra for octopus and bovine rhodopsins and their bathorhodopsin photointermediates.

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position and intensity between octopus rhodopsin and its photoproducts formed at low temperature (85 K), bathorhodopsin and isorhodopsin. The infrared difference spectra between octopus rhodopsin and octopus bathorhodopsin, octopus bathorhodopsin and octopus isorhodopsin, and octopus isorhodopsin and octopus rhodopsin are compared to analogous difference spectra for the well-studied bovine pigments, in order to understand the similarities in pigment structure and photochemical processes between the vertebrate and invertebrate systems. The structure-sensitive fingerprint region of the infrared spectra for octopus bathorhodopsin shows strong similarities to spectra of both all-trans-retinal and bovine bathorhodopsin, thus confirming chemical extraction data that suggest that octopus bathorhodopsin contains an all-trans-retinal chromophore. In contrast, we find dramatic differences in the hydrogen out-of-plane modes of the two bathorhodopsins, and in the fingerprint lines of the rhodopsins and isorhodopsins for the two pigments. These observations suggest that while the primary effect of light in the octopus rhodopsin system, as in the bovine rhodopsin system, is 11-cis/11-trans isomerization, the protein-chromophore interactions for the two systems are quite different. Finally, striking similarities and differences in infrared lines attributable to changes in amino acid residues in the opsin are found between the two pigment systems. They suggest that no carboxylic acid or tyrosine residues are affected in the initial changes of light-energy transduction in octopus rhodopsin. Comparing the amino acid sequences for octopus and bovine pigments also allows us to suggest that the carboxylic acid residues altered in the bovine transitions are Glu-122 and/or Glu-134.

Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature.

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.

Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.