Chlorophyll and pheophytin derivatives in geochemical transformation pathways: a surface-enhanced resonance Raman spectroscopic study.

Protected surface-enhanced resonance Raman spectroscopy (PSERRS) has been used to study a number of chlorophyll transformation products that have been suggested as intermediates in the so-called Treibs hypothesis which describes the transformation of ancient chlorophyll a (chl a) in the biosphere into desoxophylloerythroetio-porphyrin (DPEP) found in sedimentary environments. Both Soret- and Qy-resonant PSERR spectra have been recorded, providing two-dimensional structural fingerprints containing a number of bands which enable the presence of specific peripheral substituents to be identified. Some of these marker bands can be assigned directly to vibrational modes of the particular substituents. This has allowed further characterization of the vibrational spectrum of chl a; in particular, a vinyl mode has been identified which previously was thought to be Raman silent.

A chemometric analysis of the resonance Raman spectra of mutant carbonmonoxy-myoglobins reveals the effects of polarity.

Resonance Raman spectra of 10 carbonmonoxy-myoglobins have been obtained, including sperm whale native, pig wild-type, and the mutants H64L, H64A, V68T, V68N, H64V/V68T, F43W, F46V, and L29F. This series was chosen in order to study the effect of ligand binding pocket polarity on the positions of the v(Fe-CO) and delta (Fe-C-O) bands. Spectra of both 12CO and 13CO isotopic forms have been obtained and a detailed analysis has facilitated the identification of both the ligand-specific bands and six underlying porphyrin bands which are insensitive to this isotopic substitution. Along with a band-fitting analysis of infrared spectra, these resonance Raman data provide a comprehensive evaluation of the vibrations of the FeCO unit. The band positions of the ligand-specific modes are found to depend on the structure of the ligand binding pocket, arising from the strength of back-bonding within the FeCO unit, and clear correlations exist between the v(Fe-CO), delta (Fe-C-O), and v(C-O) band positions which characterize this synergic bonding. The results are consistent with the proposal that the vibration band positions are determined primarily by the electrostatic potential at the ligand. Five discrete band sets are observed for this set of mutants, suggesting that 5 discrete conformations occur.

Resonance Raman spectroscopy reveals novel ligation properties of the porcine myoglobin double mutant H64V/V68H.

A resonance Raman spectroscopic study of the porcine myoglobin double mutant H64V/V68H has confirmed that the ferric form is bis-histidine ligated, has revealed that the bis-histidine ligation is retained on reduction to the ferrous form, and has demonstrated that CO can displace the ligated distal histidine to produce a ferrous CO form which has a low steady-state photolability, indicating that the replacement histidine blocks the CO escape route from the binding site.

Resonance Raman spectroscopic studies of ligated mutant myoglobins.

Ligand binding (CO and N3-) to wild-type porcine myoglobin and to several mutant forms, expressed and purified from E. coli cells, has been studied using Raman spectroscopy. The v(Fe-CO) stretching vibration in MbIICO has been compared for the wild-type and mutant proteins. This gives a broad band consisting of five components, indicating five possible configurations of the bound CO. The distal pocket mutants show large variations in bandshape, the major component occurring at progressively lower wavenumber in the order: wild-type (WT) > E11 Val-->Thr (VT) > E7 His-->Val (HV) > the double mutant VT/HV (M2). Changes observed in the Raman band assigned to the azide bending mode in MbIIIN3 have been interpreted in terms of resonance structures involving two forms of azide binding. Repulsion between the bound azide ligand and the OH group of the adjacent thr residue in the VT mutant, and a shorter Fe-N(his) bond in the proximal mutant Ser-->Leu (F7), both affect this bonding. In the wild-type protein (WT), hydroxymetmyoglobin exists in a spin-state equilibrium which, at room temperature, is predominantly high-spin. In the F7 mutant this equilibrium is shifted in favour of the low-spin form. A low-spin iron species also exists in the aquometmyoglobin form of this mutant.

Ligand affinities in mutant metmyoglobins.

Ligand binding to the wild-type and a series of mutant porcine myoglobins, expressed and purified from Escherichia coli cells, has been studied using UV-VIS absorption spectroscopy. The proximal pocket mutation, F7 Ser-->Leu (F7), causes an increased affinity for OH- and N3- binding to metmyoglobin. A hydrogen bond between the F7 serine residue and the imidazole side-chain of the proximal histidine has been removed by this mutation. It is suggested that this allows the imidazole group to reorientate, reducing the steric clash between itself and the haem pyrrole nitrogen atoms and leading to a shortening of the bond between the proximal histidine and the haem iron. Other conformational changes further away from the haem pocket have also been induced, but the mutant still crystallizes under the same conditions as for the wild-type protein. A series of distal pocket mutants, E11 Val-->Thr (VT), E7 His-->Val (HV) and a mutant with both of these substitutions (M2) all have greatly reduced the OH- and N3- binding affinity. These effects have been interpreted by considering several factors: the changed stability of the aquometmyoglobin form, hydrogen-bond formation between the ligand and the E7 residue, and electrostatic repulsion between the ligand and the E11 threonine residue.

An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates.

Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C.

An intermediate redox state of cytochrome c at alkaline pH, generated upon rapid reduction by sodium dithionite, has been observed by resonance Raman (RR) spectroscopy in combination with the continuous flow technique. The RR spectrum of the intermediate state is reported for excitation both in the (alpha, beta) and the Soret optical absorption band. The spectra of the intermediate state are more like those of the stable reduced form than those of the stable oxidized form. For excitation of 514.5 nm, the most prominent indication of an intermediate state is the wave-number shift of one RR band from 1,562 cm-1 in the stable oxidized state through 1,535 cm-1 in the intermediate state to 1,544 cm-1 in the stable reduced state. For excitation at 413.1 nm, a band, present at 1,542 cm-1 in the stable reduced state but not present in the stable oxidized state, is absent in the intermediate state. We interpret the intermediate species as the state where the heme iron is reduced but the protein remains in the conformation of the oxidized state, with methionine-80 displaced as sixth ligand to the heme iron, before relaxing to the conformation of the stable reduced state, with methionine-80 returned as sixth ligand.