Spectroscopic identification of the axial ligands of cytochrome b₅₆₀ in bovine heart succinate‐ubiquinone reductase

Abstract

13 kDa), but are devoid of a b-type cytochrome [7]. The QPs Abstract The axial ligands of low potential cytochrome b560 in in SQRs from bovine heart mitochondria [8,9] and E. coli the five subunit bovine heart succinate-ubiquinone reductase com- [10,11] both contain one cytochrome b, but differ in the number plex and in the isolated quinone binding proteins have been invesand size of the constituent subunits (14-, 11-and 9-kDa subtigated using EPR and near-infrared magnetic circular dichroism spectroscopies. The results are consistent with bis-histidine ligaunits for bovine heart QPs [3,4]; 14-and 13-kDa subunits for tion with near-perpendicular imidazole rings for cytochrome b56oE. coli QPs [12,13]). Moreover, the cytochrome bs in these in the four-subunit complex. The pronounced changes in EPR enzymes differ greatly in their midpoint potentials; E m = +36 properties that accompany isolation of the cytochrome-b560 conmV and -180 mV for E. coli and bovine heart SQR, However, this was before the presence of distinct low and high Bovine heart succinate-quinone reductase (SQR) is a mempotential hemes was established in B. subtilis cytochrome b558 brane-bound component of the mitochondrial respiratory [14]. Therefore, although these results taken together attest to chain. It catalyzes the oxidation of succinate to fumarate and bis-histidyl ligation for the high potential cytochrome bs (charthe reduction of ubiquinone, thereby contributing electrons acterized by HALS EPR signals with ramped line shapes for ultimately to oxidative phosphorylation and energy transducthe low-field component), the axial ligation of the low potential tion. The purified complex can be resolved into two reconstitucytochrome bs remains ill-defined. The low potential cytotively active fractions [1,2]. One fraction, composed of the two chrome b560 of bovine heart SQR exhibits a HALS-type EPR larger subunits (70 and 27 kDa), is the soluble succinate dehysignal with a Gaussian-shaped low-field component at g = 3.46, drogenase (SDH) which contains a flavin moiety as well as but this resonance is replaced by two new resonances with low three Fe-S clusters. The other fraction, termed the quinone field g-values at 3.07 and 2.92 in purified QPs [8]. In this work, binding proteins (QPs), anchors SDH to the mitochondrial the two complementary techniques of EPR and MCD have inner membrane and comprises three smaller subunits (14, 11, been used to investigate the axial ligation of the low potential and 9 kDa) [3,4]. This membrane-bound fraction contains the cytochrome b560 in bovine heart SQR and the isolated QPs. cytochrome b560 that is the focus of this paper [5,6].As their name suggests, the QPs facilitate interaction of 2. Materials and methods SDHs and the highly homologous fumarate reductases (FRDs) with quinones. However, a specific role for the heme group in Bovine heart SQR and QPs were purified and assayed as previously mediating electron transfer to or from quinone has yet to be described [8]. Samples for spectroscopic measurements were prepar...

Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

“…The g z ϭ 2.92 peak may be due to a dramatic modification of the environment of the heme responsible for the g z ϭ 3.76 peak of the NarGHI holoenzyme, or it may be due to an altered conformation of the heme responsible for the g z ϭ 3.15 signal. EPR signals with a low-field component (g z ) at g ϭ 2.92 have been observed for conformationally modified (or "relaxed") membrane-bound b-type cytochromes (29,30). Overall, the EPR spectra of membranes enriched in NarI demonstrate that the correct insertion of both b-type hemes requires the presence of the NarGH dimer.…”

“…Attempts to rationalize such large g values within a number of membrane-bound b-type cytochromes have relied upon the assumption of distortion at the heme center controlled by the orientation of the imidazole rings (29,32). Palmer (27) has proposed that if the two imidazole-ring planes of the ligating histidines are gradually rotated from a parallel orientation, which gives a g z of about 3.0, toward a perpendicular orientation, the g z increases, and the g y and g x components of the EPR signal become progressively smaller.…”

“…In the latter case, heme insertion in the different membrane-bound subunits can only occur after the complete formation of the cytochrome bd oxidase complex. In the case of the cytochrome b 560 in bovine heart succinate-ubiquinone reductase, the pronounced changes in EPR properties that accompany isolation of the cytochrome b 560 are attributed to perturbation of the orientation of the imidazole rings of the heme bis-histidine ligands (29). In the case of the B. subtilis cytochrome b 558 , the hemes in the whole complex II and in the isolated cytochrome subunit are not in the same environment as judged by differences in the optical absorption and EPR spectra (32).…”

A structural moDAl for the membrane‐integral domain of succinate:quinone oxidoreductases

“…The single heme in mammalian and E. coli SQR and the two hemes in B. subtilis SQR have bis-histidine axial ligation as demonstrated by EPR spectroscopy combined with lowtemperature near-infrared MCD spectroscopy [13][14][15]. The ligand histidine residues have been identified in the primary sequence of the B. subtilis and E. coli SQR anchor polypeptides by site directed mutagenesis analysis [12,16,17].…”

Spectroscopic identification of the heme axial ligation of cytochrome b558 in the NADPH oxidase of porcine neutrophils

“…Perpendicular imidazole rings provide the most axial environment which leads to very intense NIR-MCD charge transfer bands (Ae values up to 560 M ~ • cm -t at 4.2 K and 5 T) and low field g-values approaching 4.0. b-Type cytochromes with properties approaching this extreme are found in membrane bound environments in which the cytochrome bridges across two membrane spanning helices, e.g. in the cytochrome bc~ complex [24] and in the membrane anchor subunits of succinate dehydrogenases [25,26]. The majority bis-histidine ligated low spin ferric heme species in cytochrome b558 is clearly in well-defined environment somewhere between the extremes of parallel and perpendicular imidazole rings.…”