We assign this faster-decaying, shorter-wavelength component with a maximum at 980 nm to Car D2 ∙+ . Although CarD2 has been proposed to be the initial electron donor in the pathway of secondary electron transfer (Lakshmi et al. 2003; Tracewell and Brudvig 2003), the specific spectral perturbations of site-directed mutations near CarD2 on the 980 nm Car∙+ species provide the first direct evidence that CarD2 is one of the redox-active Car in PSII. Previous studies have shown that the maximum of the Car∙+ near-IR peak shifts to a slightly shorter wavelength when YD is oxidized to Y D ∙ in all PSII centers (Tracewell

and Brudvig 2003). It RAD001 chemical structure was hypothesized that this was either due to an electrochromic shift caused by YD or due to biasing electron transfer so that the redox-active Car closest to Y D ∙ would remain reduced to avoid electrostatic repulsion. However, it has been observed that electrochromic shifts propagate substantial distances through PSII. For example, generating Q A − affects

the visible spectrum of BA, the accessory Chl near PA of P680, from 21 Å away, and also possibly affects the spectrum of BB, 29 Å away (Stewart et al. 2000). HSP inhibitor Although Y D ∙ would most likely have a smaller electrochromic effect than Q A – , its effects do propagate at least as far as P680 (Diner and Rappaport 2002). CarD2 is approximately 25 Å from YD. Alternatively, there are several Car cofactors in CP47 that are at a comparable or even shorter distance from YD; one Car in CP47 is 21 Å from

YD, another is 27 Å away, and two others are about 30 Å from YD. Due to closely spaced distances, an electrochromic shift would not be a definitive indicator of which Car is oxidized, even if it were observable at those distances. It is also possible that oxidation of YD may bias the path of secondary electron transfer. To pull an electron from one of the Car in CP47, two intermediate Chl∙+ would be involved that are each 20 Å from Y D ∙ , to ultimately generate a terminal Car∙+ that may be as close as 21 Å to Y D ∙ . Under these conditions, the 980 nm Car D2 ∙+ may be a more stable radical than the 999 nm Car∙+, resulting in a net shift of the Car∙+ peak to a shorter wavelengths. The near-IR Farnesyltransferase spectra of D2-G47W, D2-G47F, and D2-T50F PSII samples contain a relatively larger amount of the Chl∙+ peak as compared to the Car∙+ peak than WT PSII samples (Fig. 4B). One possibility is that the mutations around the headgroup of CarD2 caused a shift of the reduction potential of Car D2 ∙+ to a higher value, making it more difficult to oxidize CarD2 relative to other Chl and Car cofactors. This would destabilize Car D2 ∙+ , which is the predominant donor in the charge separation (980 nm Car∙+, see Fig. 5; Table 1), thus favoring Chl∙+ in a greater portion of PSII centers.