BioPHYSICAL chemistry

of Lys 296 in a hydrolysis reaction (Figure 17.5). The formation of the

Schiff base shifts the wavelength maximum from 380 to 500 nm and

produces a highly conjugated and strongly absorbing cofactor with an

extinction coefficient of 40,000 mol−^1 cm−^1.

Due to the presence of the retinal, rhodopsin could be charac-

terized by transient optical spectroscopy. In addition, the timing

problem of spectroscopic studies of many enzymes, that is the diffi-

culty in poising all of the enzymes in the same state, was easily

overcome as an incident light pulse could be used to initialize the

energy conversion. These studies showed that before the actual

isomerization occurred, the protein changes into a number of inter-

mediate states. These states were originally identified as optical

states and so are named according to the respective wavelength

maximum (Figure 17.6). Later these optical states were identified

in terms of changes in the molecular configurations. The first state,

bathorhodopsin, is formed in a few picoseconds and then converts

to a series of other states on timescales ranging from nanoseconds

to milliseconds until the all-trans-retinal is formed. The observed

changes in the optical spectra cannot be predicted based upon a

simple particle-in-a-box model (Chapter 10) as the length of the

chromophore does not change. Rather, the optical shifts can be

understood only in terms of detailed molecular models that show

changes in the energy of the ground state of the retinal, arising

from alteration of the relative position of the electron orbitals for

the carbon atoms as the bond rotates in the isomerization.

Bacteriorhodopsin

The biochemical characterization of rhodopsin proceeded slowly due

to the limited stability of the isolated protein and the irreversibil-

ity of the photoreaction. Therefore, much of the research in this

area was centered on another related protein, bacteriorhodopsin.

Bacteriorhodopsin is foundin the bacterium Halobacterium halobium,

378 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY

Schiff-base linkage

Light

11-cis isomer All-trans isomer

5 Å

11

12

11

12

Figure 17.5Upon light excitation, retinal is converted from a cisto transisomer.

Rhodopsin (500 nm)

Bathorhodopsin (543 nm)

Lumirhodopsin (497 nm)

Metarhodopsin I (480 nm)

Metarhodopsin II (380 nm)

ps Light absorption

ns

μs

ms

s

Opsin  trans-retinal

Figure 17.6The photocycle
of rhodopsin showing
the presence of many
intermediate states before
the formation of the
all-trans-retinal.