Biomedical subjects

T M Loehr

Publications and source records attributed to T M Loehr.

69 records · Page 4Linked to original sources

PubMed1983

Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue.

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.

Alcohol Dehydrogenase↗

PubMed1982

Raman spectral evidence for a mu-oxo bridge in the binuclear iron center of ribonucleotide reductase.

The Raman spectrum of the B2 subunit of Escherichia coli ribonucleotide reductase shows a peak at 496 cm-1 that appears to be in resonance with the 370-nm electronic transition of the binuclear iron center in both the native and radical-free forms of the protein. Exposure of the protein to H218O causes the peak to shift to 481 cm-1, indicating that the vibrational mode is due to an Fe-O moiety in which the oxygen can exchange with solvent. The rate of oxygen exchange (kobsd = 8.3 x 10-4 s-1) is consistent with a mu-oxo-bridged structure. Protonation of the oxygen is unlikely since the Fe-O vibration fails to shift to lower frequency in D2O. Instead, there is a gradual increase in the vibrational frequency with time to a maximum value of 502 cm-1 after 3 h in 70% with time to a maximum value of 602 cm-1 after 3 h in 70% D2O. Apparently, the deuteration of successive protein functional groups causes a slight alteration in the structure of the binuclear iron center. The resonance Raman characteristics of the Fe-O-Fe group in protein B2 are similar to those previously reported for the mu-oxo-bridged binuclear iron center in hemerythrin. A further similarity between the two proteins is the high degree of alpha-helical content. Circular dichroism measurements place this value at approximately 60% for the B2 subunit of ribonucleotide reductase.

Circular Dichroism↗

PubMed1982

Normal coordinate analysis of the copper center of azurin and the assignment of its resonance Raman spectrum.

Normal coordinate analysis that utilizes a general valence force field and the Wilson FG matrix method has been applied to several structural models representing the active site of the blue copper protein, azurin. The models included tetrahedral and square planar CuN2SS', trigonal CuN2S, and trigonal bipyramidal CuN2SS'O structures in which the Ns are imidazole nitrogens of histidines, S is the thiolate sulfur of cysteine, S' is the thioether sulfur of methionine, and O is a peptide carbonyl oxygen. For constant Cu--ligand bond lengths and initial force constants, the force field was refined against the most intense of the observed frequencies (424, 404, 369, and 261 cm-1) in the resonance Raman spectrum of Pseudomonas aeruginosa azurin. The most satisfactory fit between observed and calculated frequencies occurs for tetrahedral and trigonal structures. The calculations provide detailed assignments for the resonance Raman spectrum of azurin and reveal considerable mixing of Cu--S(Cys) and Cu--N(His) vibrational modes. The trigonal model is favored because it is shown that the approximately equal to 260-cm-1 vibration is an invariant feature in the resonance Raman spectra of blue copper proteins, even those lacking a methionine in the vicinity of the copper atom. The present analysis ascribes the high frequencies of the Cu--ligand stretching modes and the resonance enhancement to the coupled nature of their vibrations and the Franck-Condon overlaps with predominant (Cys)S leads to Cu(II) charge transfer bands in the visible region.

Azurin↗

PubMed1980

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy.

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.

Apoproteins↗

PubMed1980