Møller, I. M., Bérczi, A., van der Plas, L. H. W. and Lambers, H. 1988. Measurement of the activity and capacity of the alternative pathway in intact plant tissues: Identification of problems and possible solutions. ‐ Physiol. Plant. 72: 642–649. The cyanide‐insensitive, benzhydroxamic acid‐sensitive (e.g. salicylhydroxamic acid, SHAM) alternative pathway is located in the inner membrane of plant mitochondria and electron flow through it is not coupled to H+ pumping and ATP synthesis. When estimating the activity and capacity of the alternative pathway in intact plant tissues three main problems arise: 1) There is almost always a substantial (10–50%) KCN‐insensitive, SHAM‐insensitive residual respiration, which may be due to peroxisomal a‐oxidation of fatty acids, and which must be subtracted from all data in the further analyses. 2) There is a (KCN‐sensitive) peroxidase in many tissues that is stimulated by low SHAM concentrations (1–10 mAf), but inhibited at higher concentrations (15–50 mM). 3) High concentrations of SHAM may inhibit the cytochrome pathway. Means of identifying and alleviating these problems are presented. Provided experimental conditions are chosen such as to minimize the three problems for each new plant organ or species or each new growth condition, SHAM can be used to estimate the size of the alternatively pathway in vivo.

Significance: Cytochromes b561 (CYB561s) constitute a family of trans-membrane (TM), di-heme proteins, occurring in a variety of organs and cell types, in plants and animals, and using ascorbate (ASC) as an electron donor. CYB561s function as monodehydroascorbate reductase, regenerating ASC, and as Fe 3+ -reductases, providing reduced iron for TM transport. A CYB561-core domain is also associated with dopamine bmonooxygenase redox domains (DOMON) in ubiquitous CYBDOM proteins. In plants, CYBDOMs form large protein families. Physiological functions supported by CYB561s and CYBDOMs include stress defense, cell wall modifications, iron metabolism, tumor suppression, and various neurological processes, including memory retention. CYB561s, therefore, significantly broaden our view on the physiological roles of ASC. Recent Advances: The ubiquitous nature of CYB561s is only recently being recognized. Significant advances have been made through the study of recombinant CYB561s, revealing structural and functional properties of a unique ''two-heme four-helix'' protein configuration. In addition, the DOMON domains of CYBDOMs are suggested to contain another heme b. Critical Issues: New CYB561 proteins are still being identified, and there is a need to provide an insight and overview on the various roles of these proteins and their structural properties. Future Directions: Mutant studies will reveal in greater detail the mechanisms by which CYB561s and CYBDOMs participate in cell metabolism in plants and animals. Moreover, the availability of efficient heterologous expression systems should allow protein crystallization, more detailed (atomic-level) structural information, and insights into the intramolecular mechanism of electron transport.

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During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducible b-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochrome b561 (cyt b561) according to the wavelength maximum of its alpha-band in the reduced form. More recent evidence suggests that this protein is homologous to a b-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cyt b561 is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromes b561 probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromes b561. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cyt b561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cyt b561, compares it to the animal cyt b561, and discusses the possible physiological function of these proteins in plants.

Atomic models possessing the common structural features identified for the cytochrome b(561) (cyt b(561)) protein family are presented. A detailed and extensive sequence analysis was performed in order to identify and characterize protein sequences in this family of transmembrane electron transport proteins. According to transmembrane helix predictions, all sequences contain 6 transmembrane helices of which 2-6 are located closely in the same regions of the 26 sequences in the alignment. A mammalian ( Homo sapiens) and a plant ( Arabidopsis thaliana) sequence were selected to build 3-dimensional structures at atomic detail using molecular modeling tools. The main structural constraints included the 2 pairs of heme-ligating His residues that are fully conserved in the family and the lipid-facing sides of the helices, which were also very well conserved. The current paper proposes 3-dimensional structures which to our knowledge are the first ones for any protein in the cyt b(561) family. The highly conserved His residues anchoring the two hemes on the cytoplasmic side and noncytoplasmic side of the membrane are in all proteins located in the transmembrane helices 2, 4 and 3, 5, respectively. Several highly conserved amino acids with aromatic side chain are identified between the two heme ligation sites. These residues may constitute a putative transmembrane electron transport pathway. The present study demonstrates that the structural features in the cyt b(561) family are well conserved at both the sequence and the protein level. The central 4-helix core represents a transmembrane electron transfer architecture that is highly conserved in eukaryotic species.

POB 521, Hungary (A.B.)As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes. It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions. Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments. Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts. However, little is known about the function or subcellular localization of this unique class of membrane proteins. We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family. Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO). Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients. Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with ␣-band absorbance maximum near 561 nm. The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane. These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.Ascorbate (ASC) plays a key role in the control of growth, development, and defense responses (Davey et al., 2000; Arrigoni and De Tullio, 2002; Mittler, 2002; Pastori et al., 2003). Its role is implied in such diverse processes as the control of cell division and expansion, regulation of programmed cell death, and the regulation of the biosynthesis of ethylene and gibberellic acid. ASC biosynthetic pathways are well characterized in animals and are gradually being elucidated in plants and fungi (Smirnoff, 2001; Smirnoff et al., 2001). In contrast, the cellular mechanisms of ASC regeneration and breakdown remain poorly understood, in particular at the organelle level. The importance of ASC recycling is illustrated by the overexpression of a dehydroascorbate reductase in maize (Zea mays) and tobacco (Nicotiana tabacum), resulting in 2-to 4-fold increased levels of ASC (Chen et al., 2003).A class of membrane proteins, cytochromes b561 (Cyts b561), in plant and animal cells catalyzes transmembrane electron transfer with ASC as the electron donor, thereby contributing to ASC-mediated redox metabolism (Njus and Kelley, 1993; Asard et al., 2001). Members of this protein family use monodehydroascorbate as an electron acceptor, thereby regenerating fully reduced ASC. For example, the chromaffin granule Cyt b561 in the adrenal gland mediates intravesicular ASC regeneration, supporting the biosynt...

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Ascorbate-reducible cytochromes b561 (Cyts-b561) are a class of intrinsic trans-membrane proteins. Tonoplast Cyt-b561 (TCytb), one of the four Cyt-b561 isoforms in Arabidopsis was localized to the tonoplast. We demonstrate here that the optical spectra, EPR spectra and redox potentials of recombinant TCytb are similar to those of the well characterized bovine chromaffin granule Cyt-b561. We provide evidence for the reduction of ferric-chelates by the reduced TCytb. It is also shown that TCytb is capable of trans-membrane electron transport from intracellular ascorbate to extracellular ferric-chelates in yeast cells.