Outline of the EPR Part The nature of the EPR experiment Detection of Signals Relaxation and Saturation Phenomena The CW-EPR instrument Method of Detection Examples of EPR spectra Hyperfine coupling effects Other applications

Electron Paramagnetic Resonance Monitors paramagnetic species: transition metals and free-radicals in macromolecules. Examples: mononuclear Fe 3+, Cu 2+, Ni 3+,1+, Co 2+, RS, ROO, FMN, RCH 2, PLP Interaction with the magnetic field yields the energy separation of ground and excited states with quantum numbers m s = + ½ Transitions between the ground and excited states are induced by a microwave radiation (GHz). The energy difference is dependent on: i) the effect by the other paired electrons in the same atom which cause a spin angular momentum ii) the presence of a nuclear spin (I) which splits the spin energy levels into 2NI + 1 lines iii) the nearby presence of other paramagnetic species: coupled systems.

The Zeeman Interaction

Basics of the EPR Experiment Band  / GHz λ /cmB / mT S X Q W

Detection of Signals and Saturation At 9.45 GHz (X-band) for a signal with a linewidth of 0.1 G about spins (1 pmol) of radicals can be detected. On the other hand for Cu 2+ in solution (linewidth ~ 500 G) > 1 nmol is required. For normal free radicals EPR is at least 1000 times more sensitive than NMR.

Effect of Microwave Power and Saturation Increase in power results in signal saturation (N - ~ N + ). The spin system returns to equilibrium via the spin-lattice relaxation mechanism (rate constant = 1/T1). Spins with weak coupling to the lattice will relax slowly and are easy to saturate (trace D) with microwave power. Spin systems with strong lattice coupling are hard to saturate (trace A). The important T1 mechanism is spin-orbit coupling, strong for transition metals and weak for radicals on light atoms (N,O,S,C). Thus T1s are long for free-radicals but short for transition metals. T1s have an shallow inverse temperature dependence. Hence very low temperatures ( < 20 K) are required for the observation of paramagnetic transition metals. Lower Temperature Ideal power range

Basics of the EPR Instrument

Phase Sensitive Detection: EPR Spectra are Recorded in Derivative Mode. Effect of the Modulation Amplitude on Intensity and Resolution.

The Spin-Orbit Interaction The coupling of the unpaired electron with other paired electrons within the same atom Treated as spin-orbit: This coupling is anisotropic and gives rise to g 1 ≠ g 2 ≠ g 3 A : isotropic symmetry B and C : axial symmetry D : rhombic symmetry A : free radicals, some [Fe 3 S 4 ] 1+, Fe 3+ B: Ni 1+ C: NiFeC species, Co 2+ D: all FeS clusters, hemes, highly resolved radicals

Nuclear Hyperfine Interactions Splitting of EPR signals due to spin-nucleus interaction or spin-spin interaction (less common and better studies by DEER). Coupling constant is a tensor expressed in cm -1 (3 MHz). Since the X-axis of the spectrum is H in Gauss or mT (1mT=10G), the coupling can also be expressed in Gauss: NucleusSpin% Abundance~ A /Gauss 1H1H½ (iso) 13 C½ Mn5/ Co7/ Cu/ 65 Cu3/269.17/ Fe½2.1233

Spin nucleus interaction and quantitation by EPR Cu 2+ as acquired Single integration Double integration S=+1/2 S=-1/ For Cu 2+ (d 9 ) M I = 3/2, 4 lines are observed For Co 2+ (d 7 ) M I = 7/2, 8 lines are observed Isotopes Nuclear Spins 1 H, 15 N, 13 C, 19 F, 31 P, 57 Fe, 77 Se, 111 Cd, 113 Cd½ 2 H, 14 N 1 63 Cu, 65 Cu, 35,37 Cl3/2 17 O, 95 Mo, 97 Mo5/2 59 Co, 77 Se (7.5%)7/2

Small Molecule Case: Nitrosyl Radical Electron and nuclear spin energy levels for NO(SO 3 ) 2 2- (Fremy’s salt) in a low magnetic field.

Nitroxyl First-derivative Spectrum Spectrum of tempo in fluid solution at X-band at 20 o C.

Multi Center Examples: Iron Sulfur Clusters, High Spin Fe 3+2+

What Can We then learn from EPR? 1.Is there a paramagnetic signal? 2.What type of sample conditions produce the signal? 3.How many species are present? 4.dependence of spectra on chemical structure 5.Line shape : Dependence on physical environment – e.g., motion 6.g-value – characteristic for a chemical species 7.Spin-spin coupling – electron-nuclear coupling 8.Spin-spin coupling – electron-electron coupling 9.Relaxation times – effect of oxygen, ROS, other radicals 10.Pulse techniques 11.Biomedical imaging

Introduction to ICP-MS

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) nM ; 1-10 nM ; nM ; nM ; > 10 pM

Basic Components of an ICP-MS

Diagram of an ICP-MS; Role of CRS

Generation of Matrix and Polyatomic Species Ar, Ca, Cl and Br can be potential sources of interferences for Se and Fe. Ar, Ca, S, N, O and Cl can interfere for Ni, Co, Cu, Zn and As.

Removal of Polyatomic Species by the ORS.

Elimination of interferences with a Reaction Cell.

Application of ICP-MS to Ionomics of Yeast (A) vacuolar; (B) mitochondrial; > 2.5 RSD increases in red and > 2.5 RDS decreases in green. Eide, E.J. et.al. (2005) Gen.Biol. 6: R77.

Salient Points of ICP-MS 1. Useful for most elements which are important for Redox Biology: Se, Fe, Mn Mo, Cu, Co, Ni, Zn and (maybe) S and P 2. Since it is a form of elemental analysis, it does not distinguish redox states or chemical environments… 3. However, coupled with either GC or HPLC cam be used to identify the species which have different chemical environments (speciation) 4. Applicable to the studies of the effects of gene knockouts, knockdowns, aging, environment, diet on the ion composition within cells (ionomics) 5. Sensitivity is similar to radiolabeling without the complications of disposal, decay half lives, cost, etc.