Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time.

Photosystem II (PSII) contains two accessory chlorophylls (Chl(Z), ligated to D1-His118, and Chl(D), ligated to D2-His117), carotenoid (Car), and heme (cytochrome b(559)) cofactors that function as alternate electron donors under conditions in which the primary electron-donation pathway from the O(2)-evolving complex to P680(+) is inhibited. The photooxidation of the redox-active accessory chlorophylls and Car has been characterized by near-infrared (near-IR) absorbance, shifted-excitation Raman difference spectroscopy (SERDS), and electron paramagnetic resonance (EPR) spectroscopy over a range of cryogenic temperatures from 6 to 120 K in both Synechocystis PSII core complexes and spinach PSII membranes. The following key observations were made: (1) only one Chl(+) near-IR band is observed at 814 nm in Synechocystis PSII core complexes, which is assigned to Chl(Z)(+) based on previous spectroscopic studies of the D1-H118Q and D2-H117Q mutants [Stewart, D. H., Cua, A., Chisholm, D. A., Diner, B. A., Bocian, D. F., and Brudvig, G. W. (1998) Biochemistry 37, 10040-10046]; (2) two Chl(+) near-IR bands are observed at 817 and 850 nm in spinach PSII membranes which are formed with variable relative yields depending on the illumination temperature and are assigned to Chl(Z)(+), and Chl(D)(+), respectively; (3) the Chl and Car cation radicals have significantly different stabilities at reduced temperatures with Car(+) decaying much faster; (4) in Synechocystis PSII core complexes, Car(+) decays by recombination with Q(A)(-) and not by Chl(Z)/Chl(D) oxidation, with multiphasic kinetics that are attributed to an ensemble of protein conformers that are trapped as the protein is frozen; and (5) in spinach PSII membranes, Car(+) decays mainly by recombination with Q(A)(-), but also partly by formation of the 850 nm Chl cation radical. The greater stability of Chl(Z)(+) at low temperatures enabled us to confirm that resonance Raman bands previously assigned to Chl(Z)(+) are correctly assigned. In addition, the formation and decay of these cations provide insight into the alternate electron-donation pathways to P680(+).

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Photosystem II (PS II) contains secondary electron-transfer paths involving cytochrome b(559) (Cyt b(559)), chlorophyll (Chl), and beta-carotene (Car) that are active under conditions when oxygen evolution is blocked such as in inhibited samples or at low temperature. Intermediates of the secondary electron-transfer pathways of PS II core complexes from Synechocystis PCC 6803 and Synechococcus sp. and spinach PS II membranes have been investigated using low temperature near-IR spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. We present evidence that two spectroscopically distinct redox-active carotenoids are formed upon low-temperature illumination. The Car(+) near-IR absorption peak varies in wavelength and width as a function of illumination temperature. Also, the rate of decay during dark incubation of the Car(+) peak varies as a function of wavelength. Factor analysis indicates that there are two spectral forms of Car(+) (Car(A)(+) has an absorbance maximum of 982 nm, and Car(B)(+) has an absorbance maximum of 1027 nm) that decay at different rates. In Synechocystis PS II, we observe a shift of the Car(+) peak to shorter wavelength when oxidized tyrosine D (Y(D)*) is present in the sample that is explained by an electrostatic interaction between Y(D)* and a nearby beta-carotene that disfavors oxidation of Car(B). The sequence of electron-transfer reactions in the secondary electron-transfer pathways of PS II is discussed in terms of a hole-hopping mechanism to attain the equilibrated state of the charge separation at low temperatures.

β-carotene radicals produced in the hexagonal pores of the molecular sieve Cu(II)-MCM-41 were studied by ENDOR and visible/near IR spectroscopies. ENDOR studies showed that neutral radicals of β-carotene were produced in humid air under ambient fluorescent light. The maximum absorption wavelengths of the neutral radicals were measured and were additionally predicted by using time-dependent density functional theory (TD-DFT) calculations. An absorption peak at 750 nm, assigned to the neutral radical with a proton loss from the 4(4') position of the β-carotene radical cation in Cu(II)-MCM-41, was also observed in photosystem II (PS II) samples using near-IR spectroscopy after illumination at 20 K. This peak was previously unassigned in PS II samples. The intensity of the absorption peak at 750 nm relative to the absorption of chlorophyll radical cations and β-carotene radical cations increased with increasing pH of the PS II sample, providing further evidence that the absorption peak is due to the deprotonation of the β-carotene radical cation. Based on a consideration of possible proton acceptors that are adjacent to β-carotene molecules in photosystem II, as modeled in the X-ray crystal structure of Guskov et al. Nat. Struct. Mol. Biol. 2009, 16, 334-342, an electron-transfer pathway from a β-carotene molecule with an adjacent proton acceptor to P680•+ is proposed.

␤-Carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl Z and cytochrome b 559 in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the -carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated -electron systems and higher reduction potentials than ␤-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced ␤-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and ␤-zeacarotene (9 conjugated double bonds and 1 ␤-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the ␤-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl ؉ /Car ؉ is higher in the mutant core complexes, consistent with the higher reduction potential for Car ؉ . As the temperature increases, other carotenoids become accessible to oxidation by P 680A carotenoid molecule is an extended polyene chain with a variety of end groups and has been thought of as a "molecular wire," because, in the one-electron oxidized form, or carotenoid radical cation, the hole is delocalized over the entire conjugated -system. Carotenoid radical cations have recently been observed in several photosynthetic pigmentprotein complexes, including PS II, 3 bacterial light-harvesting complexes LHC II, and recently in plant PsbS where they may have a role in non-photochemical energy quenching (1). Among photosynthetic reaction centers, carotenoid photooxidation is unique to PS II, which uses light energy to catalyze the oxidation of water to molecular oxygen. The process of water oxidation involves highly oxidizing species that can also oxidize a carotenoid molecule. Carotenoid radical cations have been identified as intermediates in the secondary electron transport pathway of PS II (2-6).Detergent-solubilized PS II core complexes contain up to 25 different integral membrane and extrinsic polypeptide subunits, including the D1/D2 complex and light-harvesting pigment-protein complexes CP43 (PsbC) and CP47 (PsbB). Light energy is absorbed by the chlorophyll-containing light-harvesting antenna and transferred to the primary donor chlorophyll(s) coordinated by the D1/D2 polypeptides. The term "PS II reaction centers" describes a structure that includes the D1 and D2 polypeptides, cytochrome b 559 (Cyt b ...

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P 680 + . We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and β-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 nm and 814 nm and three slow-decaying bands at 810 nm, 825 nm, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl + ). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b 559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl + and Car + species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P 680 + and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulphoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P 680 + .Keywords β-carotene; carotenoid cation radical; chlorophyll cation radical; photosystem II; secondary electrontransfer pathwayIn photosystem II, light energy is transferred from the antenna Chls in the Chl-binding proteins CP43 and CP47 into the reaction center where the primary photochemical reactions are initiated. The primary electron donor Chl is most likely chlorophyll B A (see Figure 1) (1,2). The excited state of this chlorophyll drives charge separation, producing a reduced pheophytin (Pheo A − ) and the chlorophyll cation radical species, P 680 + . The charge separation, P 680 + Pheo − , is stabilized by transfer of the electron to the protein-bound primary quinone, Q A . Under normal conditions P 680 + is reduced by an electron from the oxygen-evolving complex (OEC) † This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences (DE-FG02-01ER15281), and an NIH predoctoral traineeship grant T32 GM008283 (C.A.T.). NSF Grant CHE-0215926 provided funds to purchase the ELEXSYS E500 EPR spectrometer.*To whom correspondence should be addressed. Phone: (203) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript which consists of a redox-active tyrosine, Y Z , and a tetranuclear Mn cluster (Mn 4 ) leading to the production of molecular oxygen from...

Resonance Raman (RR) spectroscopy has been used to examine the configuration of the carotenoids bound to Synechocystis PCC 6803 Photosystem II (PS II) core complexes. The excitation wavelengths used (514.5, 488.0, 476.5 and 457.9 nm) span the absorption bands of all of the approximately 12-17 neutral carotenoids in the PS II core complex. The RR spectra of the two carotenoids associated with the D1-D2 polypeptides (Car507 and Car489) of the reaction center are extracted via light versus dark difference experiments measured at 20 K. The RR results are consistent with all-trans configurations for both Car507 and Car489 and indicate that majority of the other carotenoids in the PS II core complex must also be in the all-trans configuration. The configuration of beta-carotene is relevant to its proposed function as a molecular wire in the secondary electron-transfer reactions of PS II.