K.K. Rao

Prolonged production of hydrogen gas by a chloroplast biocatalytic system.

Abstract The evolution of H2 gas in an in vitro illuminated chloroplast plus hydrogenase system was shown to function for six and a half hours at a continuous rate of about 10 μmoles H2/mg. chlorophyll/hour. Chloroplasts from various plant species, using different isolation media, and storage in a fixed state (glutaraldehyde) at 4°, −5° and −196° were shown to catalyze H2 production. Both Clostridium and E. coli hydrogenase were used. From the use of the photosystem II inhibitors DCMU and DBMIB and heat inactivation of photosystem II, it was concluded that H2O was the source of electrons for H2 gas production.

Isolation and characterization of the hydrogenase activity from the non-heterocystous cyanobacterium Spirulina maxima

In the last five years great interest has been focussed on the purification and characterization of hydrogenases from different sources, not only for their intrinsic importance, but in order to use them in the chloroplast ferredoxin-hydrogenase [l] or other suitable in vitro systems, to produce molecular Hz which is a potential fuel source. Although the enzyme activity has been found in many bacteria and algae [2], homogeneous enzyme preparations have only been isolated from photosynthetic [3-S], aerobic [6] and anaerobic bacteria [7,8]. There are a few reports about hydrogenase activity in nitrogen-fixing, heterocystous Cyanobacteria [9-121, and recently a partial purification of the enzyme from Mastigocladus laminosus [ 131, Spirulina maxima [ 131 and Anabaena cylindrica [ 141 have been achieved. Here we report the purification (to 1 lo-fold) and properties of hydrogenase activity from the nonheterocystous cyanobacterium S. maxima. This enzyme, as is the case with hydrogenases from bacterial sources [2,15], is strongly inhibited by CO and its activity is not stimulated by the presence of ATP in vitro suggesting the activity is due to a hydrogenase and not due to nitrogenase.

Electron spin–lattice relaxation of the (4Fe–4S) ferredoxin from B. stearothermophilus. Comparison with other iron proteins

The temperature dependence of the electron spin–lattice relaxation time T1 of the (4Fe–4S) ferredoxin from Bacillus stearothermophilus is studied in the range 1.2 to 40 K. This dependence is similar to that observed for the (2Fe–2S) ferredoxin from Spirulina maxima and can be interpreted with the same relaxation processes [J.P. Gayda, P. Bertrand, A. Deville, C. More, G. Roger, J.F. Gibson, and R. Cammack, Biochim. Biophys. Acta 581, 15 (1979)]. In particular, between 4 and 15 K, the data are well fitted by a second‐order Raman process involving three‐dimensional phonons, with a Debye temperature of about 60 K (45 cm−1). This would give an estimation of the highest frequency of the vibrations which can propagate through the three‐dimensional proteinic medium. In the highest temperature range (T≳30 K) the results are interpreted with an Orbach process involving an excited level of energy 120 cm−1. This process could be induced by the localized vibrations of the active site. Finally, these results are compa...

Halobacterium halobium ferredoxin A homologous protein to choroplast-type ferredoxins

A ferredoxin from Halobacterium halobium, an extreme halophile, has a typical 2Fe-2s chromophore centre and shares several other properties with chloroplast-type ferredoxins isolated from algae and plants; in particular the optical, ORD-, CDand EPR-spectra of the Halobacterium ferredoxin are very similar to the chloroplast-type ferredoxins [ 1,2]. However, it has a less negative redox potential, -350 mV, and a higher molecular weight (about 15 000) than those of chloroplast-type ferredoxins. More interestingly, it does not function in the NADP-photoreduction system of chloroplasts, though halobacterial cell-free extracts catalyse the equilibrium between ferredoxin and NADH at high salt concentrations [2]. The unique character of this ferredoxin from a bacterium led us to study its amino acid sequence and to compare it with those of other ferredoxins. We have found that the H. halobium ferredoxin molecule

Cyanidium caldarium ferredoxin: a red ag̵al type?

Chloroplast-type ferredoxin is a 2 Fe-2 S iron-sulfur protein found in blue-green algae and photosynthetic eukaryotes [ 11. A similar ferredoxin was also present in Halobacferium [2]. Recent studies on immunological cross reaction of ferredoxins showed the interrelations between blue-green and green algae or blue-green and red algae [3]. The amino acid sequences of ferredoxins from blue-green algae [4-91 and a green alga [lo] have been determined, but no other algal ferredoxin sequence is available, although a partial sequence of a red alga, Porphyra umbilicalis is known [ 111. The algal ferredoxins are evolutionarily a very diverse group judging from sequence comparisons [S,ll]. We have purified a ferredoxin from an acidothermal alga, Cyanidium caldarium which is a unicellular eukaryoticorganism of uncertain classification. This alga may represent the evolutionary transition from the prokaryotic blue-green algae to the simplest eukaryotic red alga [ 121. We describe here the amino acid sequence of Cyanidium caldarium ferredoxin and compare it with other chloroplast-type ferredoxins to assess a taxonomic and evolutionary status of Cyanidium.

The Iron-Sulphur Proteins: Evolution of a Ubiquitous Protein from Model Systems to Higher Organisms

Ferredoxins are Fe−S proteins with low molecular weight (6–12000) which act as electron carriers at very low redox potentials eg. −300 to −500 mV, in diverse biochemical processes such as bacterial and plant photosynthesis, N2 fixation, carbon metabolism, oxidative phosphorylation and steroid hydroxylation. They are found in a wide range of organisms from the ‘primitive’ obligate anaerobic bacteria, through photosynthetic bacteria, blue-green and green algae, to all higher plants and animals. Three types of ferredoxins are known −8 Fe+8 S, 4 Fe+4 S and 2 Fe+2 S. All three have been found in bacteria while the 2 Fe and some 8 Fe ferredoxins have been found in plants and animals possibly representing an evolutionary sequence. The 8 Fe ferredoxin may all be composed of two 4 Fe units. We have proposed that because of the simplicity of the 8 Fe ferredoxins (only 9 common simple amino acids in clostridia, 6 of which have been detected in the Murchison meteorite) they may have been amongst the earliest proteins formed during the origin of life. A simple peptide of about 27 amino acids could incorporate inorganic Fe+S (or possibly an existing Fe−S complex) into it nonenzymatically under anaerobic conditions to form a protein carrying one or two electrons at the potential of the H2 electrode. More than ten Fe−S model compounds have been proposed as analogues of the 4 Fe or 2 Fe containing active centres; inorganic, organometallic and peptide complexes have been synthesized. A few have many of the properties of ferredoxins but none as yet fulfills a sufficient number of criteria to substitute for ferredoxins chemically and biologically — a goal which will provide many clues to primitive peptide systems undergoing biological electron transfer reactions.

Effects of chaotropic agents on the spectroscopic properties of spinach ferredoxin

Summary The “chaotropic agents” perclorate, tricloroacetate, thiocyanate, iodide, urea and guanidine HCl were found to cause striking changes in the EPR and ORD of purified spinach ferredoxin. At low concentrations of the agents the EPR signal became sharper, with small shifts in the apparent g-values which tended to approach those of another non-sulphur protein, adrenodoxin. At higher concentrations the EPR signal changed completely in shape, though still remaining centered around g = 1.95. After a few minutes this EPR signal disappeared entirely. The agents at high concentrations also caused changes in the ORD spectrum of ferredoxin, though these did not appear to correlate directly with the EPR effects. All these changes could be reversed by removal of the agent. As the concentration of the chaotropic agents was increased the ferredoxin became more unstable, especially in the reduced state.

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins II. Two-iron ferredoxins

Variable temperature magnetic circular dichroism (MCD) spectra of a number of two-iron ferredoxins have been measured. The spectra of fully oxidised spinach and Spirulina maxima ferredoxin are independent of temperature between room temperature and 18 K, showing that no contribution to the room temperature MCD spectrum arises from the small population of low-lying excited states originating from the exchange coupling. However, the low temperature MCD spectra of the half-reduced proteins spinach and Spirulina maxima ferredoxin and adrenodoxin are all reasonably intense and temperature dependent. An interpretation of the spectrum of the charge-transfer region is suggested by starting with the assignments previously obtained from rubredoxin.

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins I. Oxidised rubredoxin

Variable temperature magnetic circular dichroism spectra have been measured on oxidised Clostridium pasteurianum rubredoxin. Evidence has been obtained for the presence of two one-electron charge-transfer transitions, sulphur to ferric ion, in the region 15 000 to 28 000 cm-1. The first moment of the lower energy band is consistent with it being the orbital transition t1 non-bonding sulphur orbital, to the 2 e ferric d-orbital. The magnitude of the spin-orbit coupling constant in the lower excited state has been determined and shown to be small compared with the axial distortion. The splitting of the low energy band observed in the absorption spectrum can therefore be equated directly with the axial distortion of the lowest excited charge-transfer state. Finally, the potential utility of making saturation experiments at very low temperatures has been examined.

Metalloproteins in the evolution of photosynthesis

Certain metalloproteins are common to all photosynthetic electron transfer chains. These include soluble proteins such as ferredoxins and cytochromes of the c2 type, and membrane-bound components such as cytochrome b, c1 and the Rieske iron-sulphur protein. The sequence of electron transfer Quinone leads to (cyt b, Fe-S, cyt c1) leads to cyt c2 indicates a common precursor to these systems and to the mitochondrial respiratory chain. In cyanobacteria the cytochrome c2 can be interchanged with the copper protein plastocyanin, and furthermore in chloroplasts of higher plants the latter is used exclusively. The ferredoxins in anaerobic photosynthetic bacteria are mostly of the [4Fe-4S] type, probably derived from those of the fermentative bacteria. These could readily be formed in the earliest cells from iron, sulphide and a very simple peptide. In the oxygen-evolving cyanobacteria and the aerobic halobacteria the [2Fe-2S] ferredoxins predominate. The electron transfer chains of the cyanobacteria have been incorporated almost unchanged into the chloroplasts of plants. The electron transfer chains of purple photosynthetic bacteria were probably the precursors of the mitochondrial respiratory chain, as shown by similarities of cytochromes c2 and succinate dehydrogenase. However a different origin of the eukaryotic cytoplasm is indicated by the presence of the copper/zinc superoxide dismutase.