BioPHYSICAL chemistry

the point where the electrons and protons are delivered. The
composition of this cofactor differs for the two types of enzyme,
containing either nickel and iron or two irons (Figure 11.7).
The arrangement of the atoms of the H cluster in the iron-only
enzyme revealed by X-ray diffraction experiments (Nicolet et al.
2000) was unexpected, showing a thiocubane iron–sulfur cluster
bridging through a sulfur of a cysteine to a surprising dinuclear
iron subcluster. The Ni–Fe cluster was found to have a remarkably
similar arrangement with a nickel atom at the site corresponding
to one of the iron positions in the H cluster.
Both clusters have coordinating cysteine residues but the
nature of the other ligands was found to be very unusual. When
the coordination was first being elucidated, the scientists were
surprised to see that the ligands did not appear to be contributed
by the surrounding protein. One of the key spectroscopic tech-
niques used to identify the ligands for the iron-only enzyme was
Fourier transform infrared spectroscopy, or FTIR (Chen et al. 2002).
The enzyme was prepared in different states: both a reduced state
and the oxidized state, prepared using thionin. The possible
contribution of CO was probed by examining the spectra in the
presence of both added^12 CO and^13 CO. The spectraof the normal
oxidized state shows infrared bands at 2086, 2072, 1971, 1948,
and 1802 cm−^1 (Figure 11.8). Upon incubation with^12 CO (actually
using naturally abundant isotopes), the infrared bands were observed at
2095, 2077, 1974, 1971, and 1810 cm−^1. When the enzyme wasincubated
with^13 CO, the infrared bands at 2017, 1974, and 1971 cm−^1 were replaced
with bands at 2000, 1971, and 1947 cm−^1 , whereas the bands at 2095, 2077,
and 1810 cm−^1 were unaffected.
The largest shifts were observed for the bands in the 1947–2017 cm−^1
region of the spectra. In the presence of^13 C, these bands shifted con-
sistently to lower wavenumbers, as would be expected, due to the
presence of the heavier isotope. The shifting arises from contributions
of two different CO ligands that are sensitive to the introduction of
CO as well as another CO that does not exchange in the presence of
added CO. Based upon additional studies, including the temperature
dependence of the spectral features, it was possible to assign the bands
in the 1947–2017 cm−^1 region to two different CO ligands. The 2017
and 1974 cm−^1 bands detected in the^12 CO samples shifted to 2000 and
1947 cm−^1 in the presence of^13 CO but the 1971 cm−^1 band is unaffected.
Due to the presence of the isotope, the other infrared bands of the
spectrum that were not associated with the exchangeable CO ligand
essentially did not change. The bands between 2072 and 2095 cm−^1
were identified as arising from a stretching mode involving another
nonprotein ligand (CN). The band at 1802–1810 cm−^1 was identified with
a bridging CO.

CHAPTER 11 VIBRATIONAL MOTION 233

O

C

FeP FeD

CysS S Y

OC

(a)

CN

CO

L

NC

[4Fe-4S]

S

X

Ni Fe

S

Cys

Cys-S

Cys-S

(b)

CN

CN

CO

Cys

S

Figure 11.7The

arrangement of the

Fe–Fe and Ni–Fe clusters

of hydrogenase with the

nonprotein ligands CO and

CN, and the unassigned

nonprotein ligands identified

as Y and L.