J. Wachtveitl et al., STRUCTURE, SPECTROSCOPIC, AND REDOX PROPERTIES OF RHODOBACTER-SPHAEROIDES REACTION CENTERS BEARING POINT MUTATIONS NEAR THE PRIMARY ELECTRON-DONOR, Biochemistry, 32(47), 1993, pp. 12875-12886
Single mutations of three amino acid residues in the vicinity of the p
rimary electron donor, P, in the reaction center (RC) from Rhodobacter
(Rb.) sphaeroides were constructed and characterized in order to stud
y the effects of hydrogen-bonding on the physical properties of P. The
mutations, Phe M197 --> Tyr, Met L248 --> Thr, and Ser L244 --> Gly,
represent single amino acid changes near P designed to introduce resid
ues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the e
ffects of nonconserved residues. The mutations were designed to change
the nonconserved H-bonding interactions of P in Rb. sphaeroides, at t
he level of a C2 acetyl, a C-9 keto, and a C-10 ester carbonyl of P, r
espectively, to those present in Rps. viridis. The Fourier transform (
pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized s
tates, from reaction centers of these mutants were studied to determin
e modifications of H-bond interactions of the pi-conjugated C2 acetyl
and C-9 keto carbonyl groups and the C-10 carbomethoxy ester carbonyl
groups of P. The vibrational spectra of reduced P in the Met L248 -->
Thr and Ser L244 --> Gly mutants reveal no evidence for changes in the
H-bonding pattern of P; this suggests that for Rb. sphaeroides wild t
ype, Ser L244 is not H-bonded to the C-10 ester carbonyl of P(L). The
vibrational spectrum of reduced P from the Phe M197 --> Tyr mutant com
pared to that of wild type can unambiguously be interpreted in terms o
f the formation of a new H-bond with an acetyl carbonyl of P, specific
ally P(M). Correlating with the new H-bond, the Phe M197 --> Tyr mutan
t exhibits an electronic absorption spectrum where the P absorption ba
nd is significantly perturbed. Intact cell and chromatophore photoblea
ching spectra of the same mutant indicate that the P absorption band h
as red-shifted by ca. 10 nm; no such behavior is observed for the othe
r mutants. As well, the P --> BPhe(L) electron transfer rate does not
seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of
P(M) to a tyrosine residue. The EPR zero-field splitting parameters, E
and D, of the primary donor triplet are only slightly modified in the
mutant reaction centers, on the order of 1%. The FT Raman spectrum of
the oxidized primary donor, P+, of the Phe M197 --> Tyr mutant shows
a 3 cm-1 upshift of the C-9 keto carbonyl band, as compared to wild ty
pe, which may indicate that the + charge is slightly more localized on
the P(L) component in P.+. Optical redox titrations of the Met L248 -
-> Thr mutant indicate that the redox midpoint potential is unchanged
(within +/- 10 mV) compared to that of wild type; the Phe M197 --> Tyr
and Ser L244 --> Gly mutants showed a small but significant increase
of +20-30 and +30-40 mV, respectively, indicating that the tyrosine-do
nated H-bond to the C2 acetyl carbonyl of P(M) in the Phe M197 --> Tyr
mutant plays no dominant role in modulating the redox properties of P
. This study suggests that the introduction of a new H-bond on the C2
acetyl carbonyl of P in Rb. sphaeroides, by a tyrosine residue at posi
tion M197, only modestly modifies its spectral properties and is not a
dominant determinant in its redox and triplet EPR properties.