Question

A coworker has just isolated a copper enzyme that catalyzes the conversion of oil sludge into soluble alcohols in the presence of O2. There are two Cu atoms per protein, which consists of a single polypeptide chain. As the bioinorganic chemist on the project, you are given unlimited quantities of the protein for the purpose of determining the active site structure. You have at your disposal a number of physical techniques, including NMR and EPR spectrometers, a magnetic susceptometer, a Mossbauer instrument, an X-ray absorption beam line, a UV-Vis spectrophotometer, a Raman/IR spectrometer, but alas, no X-ray diffractometer. You have time to complete measurements by only three techniques before you have to give a report to your colleague. Describe what measurements you would make in what order you would make them to get the most of your time, what results you might expect, and how you would use this information to characterize structurally the dicopper center. Include in your discussion the kinds of ligands that you would be looking to identify and how the methods might allow you to make such a determination.

Answer 1

Amino acids acts as ligands and get bouded to good metal ligands.

1) Electon paramagnetic resonance spectroscopy is used to study materials having unpaired electrons.

The outer electronic configuration of cu atom is 3 d¹⁰,4S¹. so cu contain 1 unpaired electron every electron has got a magnetic moment and spin quantum number m_{s= +1/2 and} m_{s= -1/2.}when an external magnetic field is applied electrons alligns parallel or antiparallel to the field with pecific energy.as there is seperation of lower and upper energy an unpaired electron can move between these levels by absobing or emitting photons.since there are two copper atoms having 1 unpaired electrons this technique can be used.

2)NMR is used to the study of three dimensional molecular structure of protein. nucleus with a spin produces a magnetic field.this magnetic field generated is the base of NMR. for identification each unique group of protons is assigned a numerical label proton and carbon chemical shifts are found from the center of ID when resolved.

3) UV specrtoscope- protein absorbs strongly at 280 nm. the peptide bonds in the amino acids also absorb at 205 nm. the UV absorbption of protein can be used to image and obtain spectra of microscopic samples. the presence of copper can also be determined in UV spectroscope.

these three techniques can be used to interpret the structure of the enzymes, the amino acids which act as the ligand and the presence of copper also can be determined.