METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION MECHANISMS OF ACTIVE SITES OFENZYMES - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 16

parameters but also followed their temperature dependencies in a wide range of
temperatures.

Two-dimentional electron spin resonance (2D ESR)
The 2D ESR technique was developed by Freed and his co-workers (Freed, 2000;
Gorcester et al., 1990; Freed and Möbius, 1992) by the analogy of the well-known NMR
two-dimentional spectroscopy, correlation spectroscopy (COSY) and spin-echo
correlation spectroscopy (SECSY) (Wütrich, 1986 and references tsherein).
In modern NMR, in order to obtain data on through-bond, scalar connectivities or
through-space, dipolar connectivities between individual spins, double or multiple
irradiation experiments are used. These rely on selective irradiation of a particular
resonance line with a radio frequency field and observation of the resulting effects in the
rest of the spectrum. With 2D ESR techniques as well as with 2D NMR techniques,
limitations of one-dimentional methods connected with overlapping resonance have been
overcome (Fig. 1.7).
Modern 2D ESR methods provide a 2D display of the homogeneous linescape across
an inhomogeneous ESR spectrum. This approach allows direct study of dynamic
processes (rotational and translational diffusion, electron transfer) and static dipole and
exchange spin-spin interactions. The possibility of the two-dimentional approach has
been significantly extended with the use Fourier transform (2D FT ESR) and electron
spin-echo (2 D SEXSY) techniques. In the 2D FT ESR spectroscopy it has become
possible to obtain a 2D display of peaks whose intensities relate directly to cross-
relaxation phenomena as spin exchange and dipole-dipole spin-spin interactions. The
ability to cover the entire spectral range of a spectrum, leads to a complete 2D mapping
of the transition rates between all points in the spectrum (two-dimentional electron-
electron double resonance, 2D ELDOR). The 2D SEXSY experiments provide a similar
map of the homogeneous transverse

spin relaxation rate and additional

information from nuclear modulation of the echo envelope.
Multiple-quantum ESR recently developed for measuring distances between spins (r)
longer than 12

is based upon double quantum coherence (DQC) pulsed ESR methods
(Freed, 2000; Borbat and Freed, 2000). Introducing an extensive cycling of four-pulse
sequence allowed the selection of the only coherence pathway related to dipole-dipole
splitting in the homogeneous ESR spectrum. The latter is directly connected to the r
value

Å

Distance estimation
In solving problems of enzyme catalysis, molecular biophysics of proteins,
biomembranes and molecular biology it is necessary to know the spatial disposition of
individual parts. One must also know the depth of immersion of paramagnetic centers in
a biological matrix, i.e. the availability of enzyme sites to substrates, distance of electron
tunneling between a donor and an acceptor group, position of a spin-label in a membrane
and in a protein globule, distribution of the electrostatic field around the PC, etc.