Heme Structure and Orientation in Single Monolayers of Cytochrome c on Polar and Nonpolar Soft Surfaces

Abstract

Polarized x-ray absorption fine structure (XAFS) spectroscopy has been performed in fluorescence mode under total external reflection conditions on frozen hydrated single monolayers of yeast cytochrome c (YCC). The protein molecules were vectorially oriented within the monolayer by tethering their naturally occurring and unique surface cysteine residues to the sulfhydryl-endgroups at the surface of a mixed organic self-assembled monolayer, itself covalently attached to an ultrapure silicon wafer. The sulfhydryl-endgroups were isolated by dilution with either methyl- or hydroxyl-endgroups, producing macroscopically nonpolar or uncharged-polar soft surfaces, respectively. Independent information on the heme-plane orientation relative to the monolayer plane was obtained experimentally via optical linear dichroism. The polarized XAFS data have been analyzed both qualitatively and by a global mapping approach limited to systematically altering the various iron-ligand distances within a model for the local atomic environment of the heme prosthetic group, and comparing the theoretically generated XAFS spectra with those obtained experimentally. A similar analysis of unpolarized XAFS data from a frozen solution of YCC was performed using either the heme environment from the NMR solution or the x-ray crystallographic data for YCC as the model structure. All resulting iron-ligand distances were then used in molecular dynamics (MD) computer simulations of YCC in these three systems to investigate the possible effects of anisotropic ligand motions on the fits of the calculated to the experimental XAFS spectra.

Linear Dichroism in the XANES of Partially Oriented Samples: Theory and Application to the Photosynthetic Manganese Complex

“…Expectedly we find that, due to the approximate C 2 -symmetries in the models used, a clear-cut discrimination between symmetry-related angles [Eqs. (18), (19) (20)- (22):…”

A Fluorescent-Photochrome Method for the Quantitative Characterization of Solid Phase Antibody Orientation

“…Equation [9] can be used to give an estimate of the depth of immersion of a fluorescent chromophore in a nonconductive matrix using experimentally measured values of the ratio k q d /k q k and known or estimated values for the lifetime of the encounter complex, c , and the parameter ␤.…”

“…Values of depth of chromophore immersion (R 0 Ϫ r V ) for different quenching mechanisms, calculated using Eq. [9] with an experimental ratio (k q d /␣ 0 k q (I)) and a lifetime of the encounter complex, c ϭ 3 ϫ 10 Ϫ10 s, are also presented in Table 2. As shown in the table, (R 0 Ϫ r V ) Ͼ 6 Å and Ͼ7.0 Å for ICHA and ET quenching mechanisms, respectively.…”

“…Among the techniques available for the characterization of the orientation of solid phase macromolecules are ellipsometry (5), circular dichroism, solid state NMR (6), coupled plasmon-waveguide resonance spectroscopy (7,8), and polarized X-ray absorption fine structure spectroscopy (9). All these methods can give an estimate of the orientation of the molecule in question as a whole, but cannot provide information on specific areas, for example, on active sites in the case of enzymes and antibodies.…”

Specific Vectorial Immobilization of Oligonucleotide‐Modified Yeast Cytochrome c on Carbon Nanotubes

“…[29] The properties of vectorially orientated YCC, chemisorbed via Cys102 at thiol-modified surfaces, have been extensively studied. [30][31][32][33] Scanning probe microscopy has shown that YCC can be immobilized directly on gold via Cys102. [34][35][36] Moreover, we have shown that YCC, vectorially chemisorbed on gold, exhibits very fast, reversible interfacial electron transfer and retains its native functionality, that is, the ability to interact with redox enzymes in solution.…”