Carotenoid analysis of halophilic archaea by resonance Raman spectroscopy.

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm(-1). The bands at 1505 cm(-1) and 1152 cm(-1) are due to in-phase C=C (nu(1) ) and C-C stretching ( nu(2) ) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH(3) groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm(-1) region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C=C stretches (1600-1500 cm(-1)), the CCH in-plane rocks (1400-1250 cm(-1)), the C-C stretches (1250-1100 cm(-1)), and the hydrogen out-of-plane wags (1000-700 cm(-1)). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry.

Activation mechanism of the CO sensor CooA. Mutational and resonance Raman spectroscopic studies.

CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum. CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism. To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays. Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO. The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact. The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket. The proximal Fe-His bond in Fe[II]CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain. A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy.

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.