RESUMEN
Chlamydomonas reinhardtii cultures, deprived of inorganic sulfur, undergo dramatic changes during adaptation to the nutrient stress [Biotechnol. Bioeng. 78 (2002) 731]. When the capacity for Photosystem II (PSII) O(2) evolution decreases below that of respiration, the culture becomes anaerobic [Plant Physiol. 122 (2000) 127]. We demonstrate that (a) the photochemical activity of PSII, monitored by in situ fluorescence, also decreases slowly during the aerobic period; (b) at the exact time of anaerobiosis, the remaining PSII activity is rapidly down regulated; and (c) electron transfer from PSII to PSI abruptly decreases at that point. Shortly thereafter, the PSII photochemical activity is partially restored, and H(2) production starts. Hydrogen production, which lasts for 3-4 days, is catalyzed by an anaerobically induced, reversible hydrogenase. While most of the reductants used directly for H(2) gas photoproduction come from water, the remaining electrons must come from endogenous substrate degradation through the NAD(P)H plastoquinone (PQ) oxido-reductase pathway. We propose that the induced hydrogenase activity provides a sink for electrons in the absence of other alternative pathways, and its operation allows the partial oxidation of intermediate photosynthetic carriers, including the PQ pool, between PSII and PSI. We conclude that the reduced state of this pool, which controls PSII photochemical activity, is one of the main factors regulating H(2) production under sulfur-deprived conditions. Residual O(2) evolved under these conditions is probably consumed mostly by the aerobic oxidation of storage products linked to mitochondrial respiratory processes involving both the cytochrome oxidase and the alternative oxidase. These functions maintain the intracellular anaerobic conditions required to keep the hydrogenase enzyme in the active, induced form.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Azufre/metabolismo , Animales , Clorofila/química , Cinética , Espectrometría de Fluorescencia , Azufre/deficienciaRESUMEN
The role of photosystem II in hydrogen photoproduction by Chlamydomonas reinhardtii cells was studied in mutants with modified D1-protein. In D1-R323D and D1-R323L mutants, the replacement of arginine by aspartate or leucine, respectively, resulted in the disruption of electron transport at the donor side of photosystem II. The rate of oxygen evolution in D1-R323D decreased twice as compared to the pseudo-wild type (pWT), and in D1-R323L no oxygen evolution was detected. The latter mutant was not capable of photoautotrophical growth. The dynamics of changes in oxygen content, the reduction of photosystem II active reaction centers (deltaF/F(1)m), and hydrogen production rate in pWT were found to be similar to the wild type if cultivated under sulfur deprivation in a closed bioreactor. The observed gradual decrease in the deltaF/F(1)m value turned to a sharp drop almost to zero followed by a partial recovery during which the production of hydrogen set in. The transition to the anaerobic phase in D1-R323D cultured in a sulfur-deprived medium occurred earlier than it happened in pWt under the same conditions. However, the partial recovery of photosystem II activity and hydrogen production started at a later time, and the rate of hydrogen production was low. The D1-R323L mutant incapable of oxygen evolution entered the rapidly anaerobiosis but produced no hydrogen. The kinetics of photoinduced redox transitions in P700 was similar in all investigated strains and was not affected by diuron addition. This implies that the mutants had a pool of reducers, which could donate electrons through the quinone pool or cytochrome to photosystem I. However, in D1-R323L mutant lacking the active photosystem II, this condition was not sufficient to support hydrogenase activity.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Mutación Puntual , Azufre/metabolismo , Proteínas Algáceas/genética , Animales , Chlamydomonas reinhardtii/genética , Transporte de Electrón/genética , Complejo de Proteína del Fotosistema II/genéticaRESUMEN
This article summarizes recent advances in the field of algal hydrogen production. Two fundamental approaches are being developed. One involves the temporal separation of the usually incompatible reactions of O(2) and H(2) production in green algae, and the second involves the use of classical genetics to increase the O(2) tolerance of the reversible hydrogenase enzyme. The economic and environmental impact of a renewable source of H(2) are also discussed.
Asunto(s)
Chlorophyta/metabolismo , Hidrógeno/metabolismo , Animales , Biotecnología/tendencias , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Oxígeno/metabolismo , Fotosíntesis , Azufre/metabolismoRESUMEN
Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O(2), which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of O2-tolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2. The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O2. Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.
RESUMEN
The [Fe]-hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. Although these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. Significant to the function of hydrogenases is how the surrounding protein structure facilitates substrate-product transfer, and protects the active site H-cluster from inactivation. To elucidate the role of protein structure in O(2) inactivation of [Fe]-hydrogenases, experimental and theoretical investigations have been performed. Molecular dynamics was used to comparatively investigate O(2) and H(2) diffusion in CpI ([Fe]-hydrogenase I from Clostridium pasteurianum). Our preliminary results suggest that H(2) diffuses more easily and freely than O(2), which is restricted to a small number of allowed pathways to and from the active site. These O(2) pathways are located in the conserved active site domain, shown experimentally to have an essential role in active site protection.
Asunto(s)
Hidrógeno/metabolismo , Oxígeno/metabolismo , Catálisis , DifusiónRESUMEN
The development of efficient biological systems for the direct photoproduction of H(2) gas from water faces several challenges, the more serious of which is the sensitivity of the H(2)-evolving enzymes (hydrogenases) to O(2), an obligatory by-product of photosynthesis. This high sensitivity is common to both FeFe and NiFe hydrogenases, and is caused by O(2) binding to their respective metallocatalytic sites. This overview describes approaches to (i) molecular engineering of algal FeFe-hydrogenase to prevent O(2) access to its catalytic site; (ii) transform a cyanobacterium with an O(2)-tolerant bacterial NiFe hydrogenase or (c) partially inactivate algal O(2)-evolution activity to create physiologically anaerobiosis and induce hydrogenase expression.
Asunto(s)
Hidrógeno/metabolismo , Animales , Dominio Catalítico , Chlamydomonas reinhardtii/enzimología , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxígeno/metabolismo , Ingeniería de ProteínasRESUMEN
The eukaryotic green alga, Chlamydomonas reinhardtii, produces H(2) under anaerobic conditions, in a reaction catalysed by an [FeFe]-hydrogenase. To identify genes that influence H(2) production in C. reinhardtii, a library of 6000 colonies on agar plates was screened with sensitive chemochromic H(2)-sensor films for clones defective in H(2) production. Two mutants of particular interest were fully characterized. One mutant, hydEF-1, is unable to assemble an active [FeFe]-hydrogenase. This is the first reported C. reinhardtii mutant that is not capable of producing any H(2). The second mutant, sta7-10, is not able to accumulate insoluble starch and has significantly lowered H(2)-photoproduction rates in comparison with the wild-type. In hydEF-1, anaerobiosis induces transcription of the two reported C. reinhardtii hydrogenase genes, HydA1 and HydA2, indicating a normal transcriptional response to anaerobiosis. In contrast, the transcription of both hydrogenase genes in sta7-10 is significantly attenuated.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Hidrogenasas/genética , Animales , Chlamydomonas reinhardtii/genética , Genes Bacterianos , MutagénesisRESUMEN
Spectrophotometric and kinetic measurements were applied to yield photosystem (PS) stoichiometries and the functional antenna size of PSI, PSII(alpha), and PSII(beta) in Zea mays chloroplasts in situ. Concentrations of PSII and PSI reaction centers were determined from the amplitude of the light-induced absorbance change at 320 and 700 nm, which reflect the photoreduction of the primary electron acceptor Q of PSII and the photooxidation of the reaction center P700 of PSI, respectively. Determination of the functional chlorophyll antenna size (N) for each photosystem was obtained from the measurement of the rate of light absorption by the respective reaction center. Under the experimental conditions employed, the rate of light absorption by each reaction center was directly proportional to the number of light-harvesting chlorophyll molecules associated with the respective photosystem. We determined N(P700) = 195, N(alpha) = 230, N(beta) = 50 for the number of chlorophyll molecules in the light-harvesting antenna of PSI, PSII(alpha), and PSII(beta), respectively. The above values were used to estimate the PSII/PSI electron-transport capacity ratio (C) in maize chloroplasts. In mesophyll chloroplasts C > 1.4, indicating that, under green actinic excitation when Chl a and Chl b molecules absorb nearly equal amounts of excitation, PSII has a capacity to turn over electrons faster than PSI. In bundle sheath chloroplasts C < 1, suggesting that such chloroplasts are not optimally poised for linear electron transport and reductant generation.
RESUMEN
The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C3 plants. Agranal bundle sheath chloroplasts (Chl a/Chl b greater than 5.0) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl a/b light-harvesting complex (LHC), and most of the cyt b559. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl a/b LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P700 are closely coordinated. Thus, it is likely that both cyt f and P700 are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II alpha from the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II beta. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.
Asunto(s)
Clorofila/metabolismo , Cloroplastos/metabolismo , Transporte de Electrón , Fotosíntesis , Zea mays/metabolismo , Sitios de Unión , Cinética , Oxidación-Reducción , FotoquímicaRESUMEN
Our previous work using the "diphenylcarbazide (DPC)-inhibition assay" has identified four amino acid (two carboxyls and two histidyls) ligands to four Mn2+ bound with high affinity on Tris-washed photosystem II (PSII) membrane fragments [Preston and Seibert (1991) Biochemistry 30, 9615-9624, 9625-9633]. One of the ligands binds a photooxidizable Mn, specifically, and the others bind either nonphotooxidizable Mn2+, Zn2+, or Co2+ [Ghirardi et al. (1996) Biochemistry 35, 1820-1828]. The current paper shows the following: (a) the high-affinity photooxidizable Mn, which donates to the oxidized primary PSII donor (YZ*), is bound to a carboxyl residue with a KM = 1.5 microM or Kd = 0.94 microM in the absence of DPC, and a Ki = 1.3 microM in the presence of DPC (both steady-state and flash approaches were used); (b) if this carboxyl is chemically modified using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), Mn2+ is photooxidized at a lower affinity (Kd = 25 microM) site that does not involve carboxyl ligands; (c) low-affinity Mn is photooxidized (possibly by YD*, the oxidized form of the alternative PSII donor) with a KM = 220 microM at a completely different site that also requires a carboxyl ligand; (d) photooxidation of high-affinity DPC by YZ* with a KM of 40-42 microM or Kd of 49-58 microM occurs at a site that does not require carboxyl residues; (e) photooxidation of low-affinity DPC with a KM = 1200 microM occurs at a site (possibly near YD) that is not affected by carboxyl modification with EDC. Due to the similarities between the binding of the high-affinity photooxidizable Mn to EDC-treated membranes and to PSII complexes from Asp170D1 mutants [Nixon and Diner (1992) Biochemistry 31, 942-948], we identify its carboxyl residue ligand as Asp170 on D1, one of the reaction-center proteins. The second carboxyl ligand identified using the DPC-inhibition assay binds Mn (but not a photooxidizable one), Zn, or Co ions. At least one of the two histidyl ligands (either His337 on D1 or another unidentified histidyl) that bind nonphotooxidizable, high-affinity Mn2+ also binds Zn2+ and Co2+.
Asunto(s)
Sustitución de Aminoácidos , Manganeso/química , Manganeso/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , 2,6-Dicloroindofenol/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Difenilcarbazida/antagonistas & inhibidores , Difenilcarbazida/metabolismo , Diurona/metabolismo , Etildimetilaminopropil Carbodiimida/farmacología , Ligandos , Oxidación-Reducción , Fotoquímica , Complejo de Proteína del Fotosistema II , Espectrometría de Fluorescencia , Spinacia oleraceaRESUMEN
In this paper, we investigate the effects of histidyl amino acid modification on high-affinity Mn binding to photosystem II (PSII) using methods similar to those used in the preceding paper [Ghirardi et al. (1998) Biochemistry 37, 0000] for carboxyl amino acid modification. Given the rather low specificity of diethyl pyrocarbonate (DEPC) for histidine modification, we modified Tris-washed PSII membranes with a novel and more specific histidyl modifier, platinum(II) (2,2':6',2"-terpyridine) chloride (Pt-TP). Both the "diphenylcarbazide (DPC)-inhibition assay" and single-turnover flash approaches were used. The concentration dependence of Pt-TP modification on steady-state measurements shows two types of interactions, each accounting for about half of the full effect. At concentrations <50 microM, Pt-TP modifies mostly histidyls and abolishes half of the observed Mn inhibition of DPC-mediated 2,6-dichlorophenolindophenol (DCIP) photoreduction (equivalent to two high-affinity, Mn-binding ligands). This effect can be blocked by addition of Mn2+ during Pt-TP modification. Double-modification experiments with DEPC and Pt-TP demonstrate that both modifiers affect the same observable histidyl residues in PSII. Above 50 microM, Pt-TP modifies mostly cysteines (or histidines in a more hydrophobic environment) and has an additional effect on the reducing side of PSII that (a) does not involve Mn binding and (b) results in the apparent abolishment of all four of the Mn-binding ligands detected by the DPC-inhibition assay. Single-flash experiments show that histidyl modification does not eliminate the binding of the high-affinity, photooxidizable Mn2+ to Asp170 on D1 (nor does it significantly affect high-affinity DPC photooxidation), but it does decrease the binding affinity (Kd) of that Mn from 0.6 to 1.5 microM, particularly at lower (<50 microM Pt-TP) concentrations. Double-modification experiments also demonstrate that the lower affinity, photooxidizable Mn-binding site, uncovered when the high-affinity site is modified with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) [see Ghirardi et al. (1998)], is not associated with a histidyl ligand. Three nonphotooxidizable, high-affinity Mn2+ ions bind to a second carboxyl and two histidyl ligands, and these Mn are not photooxidized by a flash even when the ligand to the photooxidizable Mn is modified by EDC. Proteolytic enzyme studies indicate that the two histidyl ligands identified by the DPC-inhibition assay are probably His337 on D1 and His 339 on D2, but His 332 on D1 is not eliminated.
Asunto(s)
Histidina/metabolismo , Membranas Intracelulares/metabolismo , Manganeso/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Trometamina , 2,6-Dicloroindofenol/metabolismo , Sitios de Unión , Cisteína/metabolismo , Difenilcarbazida/metabolismo , Ligandos , Cloruro de Magnesio/metabolismo , Oxidación-Reducción , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema II , Compuestos de Platino/farmacología , Unión Proteica , Spinacia oleraceaRESUMEN
The inhibition of DPC-mediated DCIP photoreduction by exogenous MnCl2 in Tris-treated photosystem II (PSII) membrane fragments has been used to probe for amino acids on the PSII reaction center proteins, including D1His337, that provide ligands for binding manganese [Preston, C., & Seibert, M. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. I, pp 925-928, Kluwer Academic Publishers, Dordrecht, The Netherlands; Preston, C., & Seibert, M. (1991) Biochemistry 30, 9615-9624 and 9625-9633]. At a concentration of 200 microM, DPC is photooxidized at both a high-affinity and a low-affinity site in PSII at approximately the same initial rate. Addition of 10 microM MnCl2 noncompetitively inhibits DPC photooxidation at the high-affinity site, with a Ki of 1.5 microM, causing a decrease of about 50% in the overall DCIP photoreduction rate. The high-affinity site for Mn binding was deconvoluted into four independent components. In earlier work, the inhibition was attributed to the tight association of either Mn2+ or Mn3+ with the PSII membrane. We report here that inhibition of DPC photooxidation may involve two different types of high-affinity, Mn-binding components: (a) one that is specific for Mn, and (b) others that bind Mn, but may also bind additional divalent cations, such as Zn and Co, that are not photooxidized by PSII. These conclusions are based on the observations that (a) DPC photooxidation can be inhibited by Zn2+ and Co2+; (b) Zn2+ and Co2+ interact with Mn2+ in a nonmutually exclusive manner, suggesting that they may share some binding components with Mn2+; (c) high-affinity Mn2+ (but not Zn2+ or Co2+) inhibition of DPC photooxidation is accompanied by nondecaying fluorescence emission, following a single saturating flash, indicating efficient electron donation by Mn2+ to YZ+; (d) Mn2+ photooxidation in the presence of DPC is not inhibited by Zn2+ or Co2+; and (e) kinetic modeling of the interaction between high-affinity Mn2+ and DPC in PSII indicates inhibition of steady-state Mn2+ photooxidation by DPC, but allows for a single photooxidation of Mn2+. We conclude that Mn inhibition of DPC photooxidation can be used to identify Mn-binding sites of physiological importance, and suggest that the Mn-specific component of the high-affinity, Mn-binding site involves the ligand to the first Mn bound during photoactivation (i.e., Asp170 on D1, as found by other investigators).
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Aminoácidos/química , Sitios de Unión , Cobalto/química , Difenilcarbazida/química , Cinética , Manganeso/química , Modelos Químicos , Estructura Molecular , Oxidación-Reducción , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema II , Spinacia oleracea , Zinc/químicaRESUMEN
A Photosystem II (PSII) reaction center particle from the stroma lamellae of Spirodela oligorrhiza has been isolated. The stroma lamellar PSII reaction center contained the same proteins found in granal PSII reaction centers, namely D1, D2, and cytochrome b559; however, the cytochrome b559 content was half of that in the granal centers. The pigment composition, 77 K fluorescence emission, and excitation spectra of the stroma lamellar reaction centers were determined. Our results indicate a fully functional PSII particle in the stromal lamellae.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Péptidos/análisis , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Complejo de Proteína del Fotosistema II , Pigmentos Biológicos/análisis , Plantas , Espectrometría de FluorescenciaRESUMEN
Peptidylprolyl cis,trans-isomerase (PPIase) activity was detected in the cytosol, mitochondria, and chloroplast of pea plants. Cyclosporin A inhibited the activity largely localized to the mitochondrial matrix while rapamycin inhibited the PPIase activity associated with the mitochondrial membranes. Differential inhibition by the two immunosuppressive drugs, the specific binding of these drugs to different mitochondrial fractions, and the immunological detection of a putative 25-kDa rapamycin-binding protein (RBP) in mitochondrial extracts attests to the presence in plant mitochondria of both cyclophilin and RBP classes of PPIases. Cyclosporin A-sensitive PPIase detected in the chloroplast was mostly localized to the thylakoids, which is suggestive of its function in the folding of membranal proteins. PPIase associated with the chloroplast stroma and the thylakoids was not inhibited by rapamycin nor was any cross-reactive RBP detected in chloroplast extracts. These results demonstrate the presence of distinct classes of PPIases in the mitochondria and the chloroplasts of plants.
Asunto(s)
Isomerasas de Aminoácido/metabolismo , Proteínas Portadoras/metabolismo , Cloroplastos/enzimología , Fabaceae/enzimología , Mitocondrias/enzimología , Plantas Medicinales , Isomerasas de Aminoácido/antagonistas & inhibidores , Isomerasas de Aminoácido/inmunología , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/inmunología , Cromatografía por Intercambio Iónico , Reacciones Cruzadas , Ciclosporina/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/metabolismo , Isomerasa de Peptidilprolil , Proteínas de Unión a TacrolimusRESUMEN
The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Oxígeno/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Animales , Chlamydomonas reinhardtii/enzimología , Hidrogenasas/metabolismo , Complejo de Proteína del Fotosistema IIRESUMEN
Two forms of the 32 kDa-D1 reaction center protein of photosystem II (PSII), having slightly different mobilities on denaturing polyacrylamide gels, have been resolved in Spirodela oligorrhiza, Glycine max L., Gossypium hirsutum L., Triticum aestivum L., and Zea mays L. The protein band with faster mobility is identified as the 32 kDa-D1 protein, and the less mobile band as a novel form, designated 32*. The two forms are structurally similar based on immunological and partial proteolytic tests. 32* is associated exclusively with the grana and is present in the PSII reaction center. Temporally, 32* appears several hours after the translocation of newly synthesized and processed 32 kDa-D1 protein from the stroma lamellae to the grana. Formation of the 32* is strictly light-dependent under physiological light intensities and correlates with a reciprocal loss of the 32-kDa form. Light induced formation of 32* is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is not coupled to linear electron transport.