EVIDENCE FOR A MIXED-LIGAND [4FE-4S] CLUSTER IN THE C14D MUTANT OF PSAC - ALTERED REDUCTION POTENTIALS AND EPR SPECTRAL PROPERTIES OF THE F-A AND F-B CLUSTERS ON REBINDING TO THE P700-F-X CORE
L. Yu et al., EVIDENCE FOR A MIXED-LIGAND [4FE-4S] CLUSTER IN THE C14D MUTANT OF PSAC - ALTERED REDUCTION POTENTIALS AND EPR SPECTRAL PROPERTIES OF THE F-A AND F-B CLUSTERS ON REBINDING TO THE P700-F-X CORE, Biochemistry, 34(24), 1995, pp. 7861-7868
PsaC-C14D (cysteine 14 replaced by aspartic acid) contains a [3Fe-4S]
and a [4Fe-4S] cluster in the F-B and F-A sites of the free protein [Y
u, L., Zhao, J., Lu, W., Bryant, D. A., and Golbeck, J. H. (1993) Bioc
hemistry 32, 8251-8258]. When PsaC-C14D is rebound to a photosystem I
(PS I) core, the g-values of 2.043, 1.939, and 1.853 appear similar to
F-A in a wild-type PS I complex [Zhao, J. D., Li, N., Warren, P. V.,
Golbeck, J. H., and Bryant, D. A. (1992) Biochemistry 31, 5093-5099].
The reconstituted PsaC-C14D-PS I complex does not contain a [3Fe-4S] c
luster; rather, a set of resonances with a rhombic line shape, a g(av)
of similar to 1.97, and broad line widths indicate the presence of a
mixed-ligand [4Fe-4S] cluster, termed F-B', in the aspartate site. Bot
h F-A and F-B' become photoreduced at 15 K, and show an interaction sp
ectrum when reduced within the same reaction center. An electrochemica
l redox study shows that F-A and F-B' titrate with midpoint potentials
near -600 mV at pH 10.0. Single-turnover flash experiments indicate t
hat F-A and F-B' function as efficient electron accepters at room temp
erature, and NADP(+) photoreduction rates are about 70% that of a reco
nstituted PsaC-PS I complex. A population of S = 3/2, [4Fe-4S] cluster
s was tentatively identified in the free PsaC-C14D protein by characte
ristic EPR resonances in the g = 5.3 region. It is proposed that P700-
F-X cores have a high affinity for PsaC-C14D that contains two cubane
clusters, and that on rebinding, the mixed-ligand cluster in the F-B'
Site crosses over from the S = 3/2 to the S = 1/2 spin state. These re
sults show that electron throughput from F-X to ferredoxin in the PsaC
-C14D-PS I complex remains efficient even though the spectroscopic and
thermodynamic properties of F-A and F-B' are altered by the presence
of the mixed-ligand iron-sulfur cluster in the F-B site.