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Infrared spectroscopy has been used to examine the oxidized and CO-inhibited forms of Fe-only hydrogenase I from Clostridium pasteurianum. For the oxidized enzyme, five bands are detected in the infrared spectral region between 2100 and 1800 cm(-1). The pattern of infrared bands is consistent with the presence of two terminally coordinated carbon monoxide molecules, two terminally coordinated cyanide molecules, and one bridging carbon monoxide molecule, ligated to the Fe atoms of the active site [2Fe] subcluster. Infrared spectra of the carbon monoxide-inhibited state, prepared using both natural abundance CO and 13CO, indicate that the two terminally coordinated CO ligands that are intrinsic to the enzyme are coordinated to different Fe atoms of the active site [2Fe] subcluster. Irradiation of the CO-inhibited state at cryogenic temperatures gives rise to two species with dramatically different infrared spectra. The first species has an infrared spectrum identical to the spectrum of the oxidized enzyme, and can be assigned as arising from the photolysis of the exogenous CO from the active site. This species, which has been observed in X-ray crystallographic measurements [Lemon, B. J., and Peters, J. W. (2000) J. Am. Chem. Soc. 122, 3793], decays above 150 K. The second light-induced species decays above 80 K and is characterized by loss of the infrared band associated with the Fe bridging CO at 1809 cm(-1). Potential models for the second photolysis event are discussed.

An infrared-detectable group senses changes in charge density on the nickel center in hydrogenase from Chromatium vinosum Bagley, K.A.; Duin, E.L.; Roseboom, W.; Albracht, S.P.J.; Woodruff, W.H. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACT: 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(C0)) in the Ni(I1)CO species of this enzyme

Hydrogenases catalyze the reversible splitting of dihydrogen (H 2 7 2H ϩ ϩ 2e Ϫ ) and are common in many microorganisms. Their physiological role is either to acquire reducing equivalents from H 2 or to dispose of excess reducing equivalents from fermentation via the reduction of protons. Hydrogenases are often intimately complexed to modules containing other redox proteins. In this way the metabolism of dihydrogen is linked to redox chemistry with a wide variety of electron acceptors and donors like NAD(P)(H), b-and c-type cytochromes, factor F 420 , S 2Ϫ , and membrane-bound (mena)quinones. With regard to the overall metal content three classes of hydrogenases can be discriminated. The majority of hydrogenases contain nickel in addition to iron and are termed [NiFe]-hydrogenases. The minimal protein unit required for activity contains two subunits, a large one (46 -72 kDa) and a small one (23-38 kDa; for review see Ref. 1). The three-dimensional structure of the enzyme from Desulfovibrio gigas disclosed (2, 3) that the active site is a Ni-Fe dinuclear center attached to the large subunit via four thiolates from Cys residues. The iron atom has three non-protein ligands, with an electron density equivalent to diatomic molecules. The small subunit contains two [4Fe-4S] clusters and one [3Fe-4S] cluster. From a comparison of the amino acid sequences of [NiFe]-hydrogenases, it can be concluded that only the cubane cluster closest to the active site is conserved in all enzymes (1). FTIR 1 studies (4 -6) showed that [NiFe]-hydrogenases contain a set of three infrared absorption bands in the 2100 to 1850 cm Ϫ1 spectral region, not found in any other proteins. As the frequency of these bands is very sensitive to the status of the active site, it was concluded that they are due to intrinsic ligands (diatomic molecules with a triple bond or triatomic molecules with two adjacent double bonds) of the active site. Also a unique lone low spin Fe(II) site was detected, in addition to the high spin iron sites of the Fe-S clusters, by Mössbauer spectroscopy (7).A second class forms the [Fe]-hydrogenases (for review see Ref. 8); no other metal than iron is present in these enzymes. The prosthetic groups are located in only one subunit and minimally consist of two classical [4Fe-4S] clusters and a hydrogen-activating site, called the H cluster. The latter active site was speculated to be an Fe-S cluster with 4 -7 iron atoms (9, 10). It was recently discovered (6) that also [Fe]-hydrogenases show FTIR bands in the 2100 to 1850 cm Ϫ1 spectral region, which strongly shift upon changes of the redox state of the enzyme. Hence a similar architecture was suggested for the active sites of [NiFe]-and [Fe]-hydrogenases.The third class of hydrogenases does not contain any metal and occurs in methanogenic Archaea (11, 12). These enzymes, H 2 -forming N 5 ,N 10 -methylenetetrahydromethanopterine dehydrogenases, can activate H 2 only in the presence of their second substrate.In this paper we present spectroscopic as well as chemical evidence t...

Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes. in the chro-*mophore and apoprotein 'that change in intensity or position between light-adapted bacteriorhodopsin and the K and M-intermediates in its photocycle and between dark-adapted and lightadapted bacteriorhodopsin. Our infrared' measurements provide independent verification of resonance Raman results that in lightadapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is un-,protonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most'likely interpretation of deuterium shifts of the chromophore hydrogen out-ofplane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of.those in light-adapted and'darkadapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.Bacteriorhodopsin (bR) is the light-energy transducing protein found in the purple membrane (PM) of the extreme halophile Halobacterium halobium (1-4). The chromophore in bacteriorhodopsin is a single molecule of retinal, covalently bound to the £-amino group of a lysine (Lys-216) via a Schiffbase linkage (Fig 1). Upon absorption of light, the light-adapted form of bR (bR ) undergoes a photocycle, bRLA +--* K --L --M -O 0 bRLA, during which protons are pumped from the inside of the cell to the extracellular medium. The resulting proton gradient is used by the cell to generate chemical energy in the form ofATP and drive other energy-requiring processes. In the dark, bRLA thermally converts to the dark-adapted form of bR (bRDA).The mechanism of this light driven proton pump has been studied by using visible and ultraviolet, resonance Raman (5), and infrared (IR) (6-8) spectroscopies and chemical extraction techniques. These investigations strongly suggest that during the photocycle changes occur in both the isomeric state of the chromophore and the state ofprotonation of the Schiff'base. In particular, chemical extraction experiments have provided evidence that the chromophore in bRLA is in an all-trans configuration, that in the L and M states it is in a 13-cis configuration, and that in bRDA the chromophore exists in two isomeric forms, all-trans and 13-cis, in a ratio of approximately 1: 1 (9-11).Evidence for the conformation of the chromophore in situ comes primarily from comparisons between the resonance Raman vibrational spectra in both 'H20 and 2H20 of native bR, bR in which analogs ofretinal have been incorporated, and retinal 'Schiff bases. Analysis of the results from such work is dif-CH3 CH3

, W. H. (1994). Infrared studies on the interaction of carbon monoxide with divalent nickel in hydrogenase from Chromatium vinosum. Biochemistry, 33,[9229][9230][9231][9232][9233][9234][9235][9236]

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.

Light-induced metastable linkage isomers of trans-[Ru(NH(3))(4)Cl(SO(2))]Cl and trans-[Ru(NH(3))(4)(H(2)O)(SO(2))](C(6)H(5)SO(3))(2) have been identified for the first time using photocrystallographic methods. In both linkage isomers the SO(2) ligand is side bound, but the Ru-O and Ru-S distances are considerably longer and almost equal in the trans-H(2)O isomer. DFT calculations confirm that both isomers correspond to minima on the ground-state potential energy surface and also predict the existence of a second oxygen-bound isomer for both compounds. The decay of the light-induced species has been studied by both DSC and IR. Activation energies for the thermal back-reaction, as derived from the temperature-dependent disappearance of light-induced IR bands, are 50.0 and 58.4 kJ/mol for the two isomers, which is larger than the corresponding numbers for photoinduced side-bound nitrosyl linkage isomers.