Christopher J. Bender

Cockroach transferrin closely resembles vertebrate transferrins in its metal ion-binding properties: A spectroscopic study

The optical and electron paramagnetic resonance (EPR) spectroscopic properties of a transferrin from the cockroach Blaberus discoidalis have been investigated to determine the relation of this protein to vertebrate transferrins. Difference spectrophotometry substantiates the involvement of tyrosyl residues in iron binding, and confirms the specific binding of two equivalents of iron per molecule. The far-UV CD spectrum also indicates a secondary structure with marked similarity to those of vertebrate transferrins. EPR studies show a dependence of iron binding on (bi)carbonate, consistent with the absolute requirement of transferrins for a synergistic anion in binding iron. Continuous wave (CW) and pulsed EPR studies of the cupric complex of the protein implicate a histidyl nitrogen ligand in metal coordination, as in human transferrin. Additional studies establish that the pH-dependent release of iron is similar to that of human serum transferrin. The present data confirm cockroach transferrin as an authentic member of the transferrin superfamily, thereby suggesting an ancestral relationship of insect to vertebrate transferrins.

Continuous wave electron nuclear double resonance spectroscopy.

Publisher Summary The rapidly accelerating progress in the biological applications of electron nuclear double resonance (ENDOR) spectroscopy seen since the 1970s reflects a combination of instrumental and conceptual advances. Structural information and its functional implications have been wrested from ENDOR studies of almost all magnetic nuclei coupled to the paramagnetic centers encountered in biological molecules. ENDOR spectroscopy, with the detailed insight it offers into the local structure of a paramagnetic centers in solution and the alterations of such structure during biological function, can often complement the tremendous power of X-ray crystallography in studies of metalloproteins. Interpretation of ENDOR spectra merges intuition with analytic rigor with the limitations of the one, often surmounted by the power of the other. Sensitivity problems persist but are yielding to studies at ever higher frequencies. Fruitful exploitation of Triple resonance in studying metalloproteins has yet to be accomplished. The wide availability of the personal computer, with its dramatically increasing speed and power, has enormously facilitated processing and interpretation of spectroscopic data. A persistent difficulty is to achieve understanding of the relaxation processes and pathways governing ENDOR spectroscopy; such understanding has yet to attain the peaks achieved by other, perhaps simpler, spectoscopies. The ingenuity and resourcefulness of experimenters remain the most powerful tools in the armamentarium of ENDOR spectroscopy.

Electron spin-echo modulation spectroscopic study of the type I copper center associated with stellacyanin from Rhusvernicifera

The type I copper protein stellacyanin was examined by electron spin-echo modulation (ESEEM) spectroscopy in order to test the multi-frequency approach of analyzing weak I = 1 hyperfine interactions when two slightly dissimilar contributions to the echo modulation interferogram are present. Two distinct contributions to the stellacyanin spectrum are resolved by the multi-frequency acquisition of ESEEM spectra, and the graphical approach to their deconvolution is described. The stellacyanin ESEEM spectrum can be completely explained in terms of two imino nitrogen contributions assigned to the histidine side chain ligands to copper whose ring orientations are perpendicular to one another (minimally 60°). The spin Hamiltonian parameters assigned to these two nitrogen atoms are: e2Qq = 1.45 MHz, η = 0.9, A = 2.0 MHz; and e2Qq = 1.50 MHz, η = 0.9, A = 1.5 MHz. Analysis of the double quantum transition lineshape indicates that the nuclear hyperfine interaction tensor is very nearly isotropic. There is no evidence in the data reported here for the detection of an amino nitrogen interaction that would arise from the predicted ligand GlnNe.

Electron spin echo modulation study of the Type I copper protein rusticyanin and its mutant variant His85Ala

Electron spin echo modulation (ESEEM) spectra were recorded at 500 MHz intervals for the Type I protein rusticyanin and its engineered variant His85Ala, which lacks one of the two imidazole ligands to copper. The Zeeman and g-value dependence of the spectral peaks were systematically mapped and accurately predicted that low intensity lines in the wild type (w.t.) spectrum were combination lines, as verified in the His85Ala spectra. Analysis reveals that the w.t. ESEEM spectra are attributable to a pair of HisNe only, and the NQI parameters e2Qq and η are 1.37 ± 0.03 MHz and 0.90 ± 0.05 for both HisNe in the w.t. protein. The nuclear hyperfine interaction is estimated as 1.8 MHz, and includes a dipolar interaction of 0.5 MHz. The principal difference between the two imidazole nitrogen couplings is manifest in the dispersion of the combination lines, which suggests non-equivalent orientations of the respective superhyperfine tensors (or tilt of the imidazole ring). The protocol demonstrates a general procedure for accurately determining superhyperfine parameters from powder ESEEM spectra. Finally, peak shifts among ESEEM spectra of His85Ala–X (X = H2O, Cl−, Br−) demonstrate that the imidazole ligand powder superhyperfine spectrum is sensitive to chemical effects in the metals ligand sphere, thus suggesting that the method may be used for evaluation in molecular design.

Histidines 138 and 143 are Copper Binding Ligands in Chromobacterium Violaceum Phenylalanine Hydroxylase

Phenylalanine hydroxylase (PAH) from Chromobacterium violaceum (CV) is known to bind an equivalent of divalent copper.1 Studies using pulsed EPR spectroscopy2 have suggested that there are two equatorial imidazoles coordinated to Cu(II) of CV PAH. This observation has been supported by copper-histidine model complexes of the active site3 and more recently by X-ray absorption spectroscopy of the copper containing enzyme.4 We have used a combination of site directed mutagenesis and pulsed EPR spectroscopy to probe the copper binding site of CV PAH and identify the Cu(II) ligands.