Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

Abstract

We have established a new purification procedure of cytochrome b 561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b 561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at g z ‫؍‬ 3.14 and g z ‫؍‬ 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at g z ‫؍‬ 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (b L or b 566 ) of the mitochondrial complex III, indicating that the g z ‫؍‬ 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid-and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…Only CYBASC1 of wild watermelon was found targeted to the PM in GFP construct experiments with onion epidermal cells (Nanasato et al, 2005). Moreover, the optical spectra of bona fide cytochrome b561 (CYBASC1 in plants; Tsubaki et al, 1997;Bérczi et al, 2007) are characterized by an asymmetric a-band with a maximum close to 561 nm and a shoulder at 557 nm, unlike the cytochrome involved in trans-PM electron transport whose a-band is definitely symmetric (Asard et al, 1992). Overall, it is unlikely that cytochrome b561 (CYBASC1) represents a significant fraction of high-potential cytochromes b of plant PM.…”

“…Cytochromes b561 are high-potential, transmembrane redox proteins of about 25 kD made of six membrane-spanning a-helices, which bind two hemes b. One heme is predicted to be close to an ascorbate binding site facing the cytosol, whereas the second heme faces the opposite side of the membrane and can be oxidized by either MDA or ferrichelates (Tsubaki et al, 1997;McKie et al, 2001;Bérczi et al, 2005;Kamensky et al, 2007). Plants contain several orthologous genes to animal cytochrome b561 Bashtovyy et al, 2003).…”

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

“…It has been observed that recombinant and native plant and mammalian Cyts b 561 have characteristic Asc-reducible spectra (30,33,(41)(42)(43)(44). To confirm this biochemical characteristic for DCytb, the absorption spectra were measured using stripped membrane fractions from yeast cells expressing mouse DCytb (Fig.…”

Redox enzymes in the plant plasma membrane and their possible roles

“…This cytochrome b is very similar to the chromaffin granule cytochrome b 561 ; not only the biochemical and spectrophotometric characteristics and the rather high redox potential values but also the transmembrane electron transporting capabilities of the two cytochromes b were similar (Asard, Horemans & Caubergs 1992, 1995a . On the basis of multiple alignment analysis and electron spin resonance measurements (Tsubaki et al 1997) it seems possible that the chromaffin granule cyt. b 561 has six trans-membrane segments, two distinct hemes and binding sites on opposite sides of the membrane for ascorbate and MDA.…”