RESUMEN
We present here the life and the work of Paul A. Castelfranco (1921-2021), a very special person who was not only a top chemist of chlorophyll biosynthesis, but also made major contributions on fatty acid oxidation, acetate metabolism and cellular organization. He led an extraordinary and exemplary life as a human being. We present here both his personal life as well as his scientific life, which is followed by reminiscences by William Breidenbach, Kevin Smith, Alan Stemler, Ann Castelfranco, and John Castelfranco. As the subtitle of this Tribute implies, till the end Paul was a scientist par excellence, an intellectual with unlimited curiosity, a humanist, and a man of enduring religious faith. We all miss him dearly.
RESUMEN
This study provides evidence that, in the soluble fraction from buffer-washed pea thylakoids, one form of soluble carbonic anhydrase (CA) is associated with rubisco in a stromal protein complex. On native-PAGE gels, it is present as a protein band with MW approximately 160 kDa. On SDS-PAGE gels, it is resolved as a single 25-kDa polypeptide. Analysis of Western blots developed with polyclonal antibodies to barley rubisco and to soluble pea CA shows that a 160-kDa protein with CA activity is associated with rubisco in a protein complex localized on the outer surface of thylakoid membranes.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Pisum sativum/enzimología , Proteínas de Plantas/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Tilacoides/enzimología , Anhidrasas Carbónicas/química , Electroforesis en Gel de Poliacrilamida , Membranas Intracelulares/enzimología , Peso Molecular , Proteínas de Plantas/químicaRESUMEN
Peroxydicarbonic acid (Podca), a proposed intermediate in photosynthetic oxygen evolution, was synthesized electrochemically. Consistent with literature descriptions of this compound, it was shown to be a highly reactive molecule, spontaneously hydrolyzed to H2O2, as well as susceptible to oxidative and reductive decomposition. In the presence of Mn2+ or Co2+, Podca was quickly broken down with release of O2. The liberation of O2, however, was partially suppressed at high O2 concentrations. In the presence of Ca-washed photosystem II-enriched membranes lacking extrinsic proteins, Podca was decomposed with the release of O2, but only under conditions favoring photosynthetic electron flow (light plus a Hill oxidant). A model is proposed that details how peroxydicarbonic acid could act as an oxygen-evolving intermediate. The hypothesis is consistent with the well-established Kok model and with recent findings related to the chemistry of oxygen evolution.
Asunto(s)
Oxígeno/metabolismo , Ácido Peracético/metabolismo , Fotosíntesis/fisiología , Bicarbonatos/química , Bicarbonatos/metabolismo , Anhidrasas Carbónicas/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Manganeso/metabolismo , Modelos Biológicos , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
The effects of Cl(-), Mn(2+), Ca(2+), and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl(-), and strongly inhibited above this concentration. This enzyme is activated by Mn(2+) and stimulated somewhat by Ca(2+). The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl(-) up to 0.4 M. Ca(2+) appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn(4) complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Cationes Bivalentes/farmacología , Cloruros/farmacología , Membranas Intracelulares/efectos de los fármacos , Complejo de Proteína del Fotosistema II/metabolismo , Anhidrasas Carbónicas/análisis , Anhidrasas Carbónicas/clasificación , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Pisum sativum/citología , Complejo de Proteína del Fotosistema II/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Tilacoides/química , Zea mays/citologíaRESUMEN
The purpose of this study was to identify the location of one of the two sources of carbonic anhydrase (CA) activity associated with the PSII complex in chloroplast membranes. We tested the hypothesis that the extrinsic 33 kDa protein, OEC33, associated with the oxygen-evolving complex (OEC), is one source of CA activity. We found that precursor OEC33 expressed in Escherichia coli exhibits CA activity, but the expressed precursors of OEC24 or OEC17 do not. The CA activity of OEC33 remained after treatment at 90 degrees C for 15 min. Additional biochemical evidence supports the hypothesis. Only those wash treatments that remove the OEC33 from PSII also remove CA activity. Both immunoblot and CA activity show that the CA tracks the OEC33, in parallel, when PSII undergoes washing at different CaCl2 concentrations. The OEC33 protein purified by HiTrap Q anion exchange chromatography has CA activity that is inhibited by an antibody against OEC33. PSII membranes washed with 1 M CaCl2 to remove OEC33 can be reconstituted either with extracted, purified, OEC33 or with the E. coli-expressed precursor OEC33. Reconstitution partially restores both oxygen evolution and CA activity. For maximal CA activity, OEC33 requires manganese as a cofactor.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Pisum sativum/enzimología , Proteínas de Plantas/metabolismo , Anticuerpos/inmunología , Cloruro de Calcio/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/fisiología , Cloroplastos/efectos de los fármacos , Cloroplastos/enzimología , Cloroplastos/fisiología , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Estabilidad de Enzimas , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Calor , Oxígeno/metabolismo , Pisum sativum/química , Pisum sativum/genética , Pisum sativum/metabolismo , Complejo de Proteína del Fotosistema II/química , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
One form of carbonic anhydrase (CA) has been observed in maize (Zea mays) thylakoids and photosystem II (PSII)-enriched membranes. Here, we show that an antibody produced against a thylakoid lumen-targeted CA found in Chlamydomonas reinhardtii reacts with a single 33-kD polypeptide in maize thylakoids. With immunoblot analysis, we found that this single polypeptide could be identified only in mesophyll thylakoids and derived PSII membranes, but not in bundle sheath thylakoids. Likewise, a CA activity assay confirmed a large amount of activity in mesophyll, but not in bundle sheath membranes. Immunoblot analysis and CA activity assay showed that the maximum CA can be obtained in the supernatant of the PSII-enriched membranes washed with 1 M CaCl(2), the same procedure used to remove all extrinsic lumenal proteins from PSII. Because this CA reacts with an antibody to lumen-directed CA in C. reinhardtii, and because it can be removed with 1 M CaCl(2) wash, we refer to it tentatively as extrinsic CA. This is to distinguish it from another form of CA activity tightly bound to PSII membranes that remains after CaCl(2) wash, which has been described previously. The function of extrinsic CA is not clear. It is unlikely to have the same function as the cytoplasmic CA, which has been proposed to increase the HCO(-)(3) concentration for phosphoenolpyruvate carboxylase and the C(4) pathway. We suggest that because the extrinsic CA is associated only with thylakoids doing linear electron flow, it could function to produce the CO(2) or HCO(-)(3) needed for PSII activity.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Cloroplastos/enzimología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Hojas de la Planta/enzimología , Zea mays/enzimología , Animales , Cloruro de Calcio/farmacología , Chlamydomonas reinhardtii/enzimología , Clorofila/análisis , Cloroplastos/efectos de los fármacos , Immunoblotting , Complejos de Proteína Captadores de Luz , Consumo de Oxígeno , Complejo de Proteína del Fotosistema II , Hojas de la Planta/efectos de los fármacos , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacología , Tilacoides/efectos de los fármacos , Tilacoides/enzimología , Zea mays/efectos de los fármacosRESUMEN
A short list of the twentieth century's dominant figures in photosynthesis would unquestionably include Otto Warburg. One of his many discoveries, the 'bicarbonate effect' remains a lasting puzzle to his heirs in the field. Recent developments in this area of research have renewed interest and call for a re-examination of the ideas surrounding this controversial topic. Focus here will be on hypotheses developed by a small number of researchers who proposed that bicarbonate may be involved in oxygen evolution. The effect of bicarbonate on the acceptor side of Photosystem II (PS II) is discussed by Jack van Rensen (in this issue).