XAFS on Vectorially-Oriented Single Monolayer Protein Samples

Abstract
Polarized XAFS measurements were carried out on a single monolayer of cytochrome c tethered to a self assembled organic monolayer. The monolayer contains 10¹² to 10¹³ protein molecules per cm², which is 10 to 100 times more dilute than a single monolayer of small inorganic molecules. Thus, the spectra were collected under total reflection conditions to ensure a reasonable signal to noise ratio by increasing signal counts and, in the mean time, reducing background. The.spectrum with X-ray polarization parallel to the substrate surface is found to be similar to the spectrum'of the perpendicular orientation. However, the spectra are somewhat different from the spectrum of cytochrome c in a frozen solution. The differences may be attributed to thermal effects and/or the impurity signal from the silicon or quartz substrates. Optical linear dichroism measurements determined the tilt angle between the average heme-plane and the substrate surface plane to be about 40°, consistent with XAFS observations. XAFS experiments on a single protein monolayer provide new opportunities for probing metal centers in biology.