800 MHz ¹H NMR solution structure refinement of oxidized cytochrome c₇ from Desulfuromonas acetoxidans

Abstract

The solution structure of Desulfuromonas acetoxidans cytochrome c 7 has been refined by using 1 H-NMR spectra recorded at 800 MHz and by using pseudocontact shifts in the final energy minimization procedure. The protein, composed of 68 amino acids, contains three paramagnetic heme moieties, each with one unpaired electron. The largely distributed paramagnetism broadens the lines in several protein parts. The structure is now relatively well resolved all over the backbone by the use of 1315 meaningful NOEs and 90 pseudocontact shifts. The statistical analysis of the structure indicates its satisfactory quality. The protein-fold is quite similar to that of the analogous four-heme cytochromes c 3 for those parts which can be considered homologous. The solvent accessibility and the electrostatic potential surfaces surrounding the three hemes have been analyzed in terms of their reduction potentials. The resulting magnetic susceptibility anisotropy data obtained from pseudocontact shifts are analyzed in terms of structural data.Keywords : NMR; multiheme cytochrome; solution structure ; magnetic susceptibility tensor. Desulfuromonas acetoxidans is a bacterium which produces energy via a cytochrome-linked electron-transfer process to either sulfur or fumarate, which act as electron acceptors [12]. This organism produces a cytochrome, cytochrome c 7 (cyt c 7 , hereafter) which, in the oxidized form, contains three low-spin heme groups, each with one unpaired electron [13]. The heme iron ions are bound to histidine residues from analogy with cytochrome c 3 from Desulfovibrio organisms, which have four hemes and which show large similarity with the present system [14Ϫ21]. The function of cyt c 7 has not yet been elucidated; however, it has been reported that it could function as a terminal reductase [22] and more recently that it acts as a polysulfide reductase [23]. It could represent the terminal reductase in D. acetoxidans. However, its physiological electron donor still remains to be identified.The X-ray structure of cyt c 7 is not available. From 600-MHz 1 H-NOESY, COSY, and TOCSY experiments a structure was obtained although only two ring protons of two of the six axial histidine rings could be assigned, being as broad as 500Ϫ 1000 Hz [24]. No signal of the other four histidine rings was assigned nor has been assigned now. Furthermore, isotope labeling cannot be accomplished as, despite cyt c 7 being also expressed in Desulfovibrio desulfuricans [25], the growth in minimal medium of both bacteria is very poor and very large volumes (of the ordered of several hundreds liters) are needed to prepare an NMR sample. The structure of the protein backbone is such that not many long-range NOEs could be expected and short-range NOE constraints are not best suited to solve this type of structure. Despite these difficulties a family of 20 conformers was obtained with the program DIANA [26] with small violations and a backbone rmsd of 0.70Ϯ0.22 Å with respect to the average structure [24]. The extensive use of the REDAC strateg...

The metal reductase activity of some multiheme cytochromes c : NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c 7

“…1 is dipolar in origin and that the dominant correlation time for the interaction is the rotational correlation time of the protein estimated from the StokesEinstein equation. The 35 NMR solution structures of fully oxidized Cyt c 7 (12) were annealed in 10,000 steps with the program DYANA (25) by adding the chromium-proton distance constraints to the other available constraints (12). (ii) The metal-proton distances thus obtained in the structural model were plotted against (⌬ ) Ϫ1͞6 .…”

“…§ The three heme-containing cytochrome c 7 from the sulfurreducing bacterium Desulfuromonas acetoxidans (Cyt c 7 hereafter) has been proposed to have a role as electron-transfer protein in the sulfur metabolism of this bacterium, acting as a terminal reductase in the metabolic pathway by directly reducing elemental sulfur to sulfide (9); it has been suggested also that it could be involved in the reduction of iron(III) and manganese(IV) (10). The solution structures of the fully oxidized and fully reduced species are available, followed by the x-ray structure of the oxidized species (11)(12)(13)(14). The availability of the assigned NMR spectra and the nuclear Overhauser effects (NOEs; refs.…”

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12nm protein with interacting hemes

“…The cytochrome c 7 is a small protein (70-75 amino acids) with three hemes covalently attached to the polypeptide through the CXXCH motif, and each heme has bis-His axial ligation. The hemes are named I, III and IV due to their structural homology to the equivalent hemes of tetraheme cytochromes c 3 (Assfalg et al, 1998). Three larger multiheme proteins 2 (two containing 12 hemes, GSU1996 and GSU0592, and one with 27 hemes, GSU2210) are also encoded in the Gs genome (Pokkuluri et al, 2004a).…”

Nuclear Magnetic Resonance as a Tool to Characterize the Interactome of Heme Proteins

“…[44][45][46][47] Later, the measure of PCSs for tens of nuclei (including 15 N for backbone amides) has allowed an accurate estimate of the magnetic anisotropy parameters in deoxymyoglobin and cytochrome c′ (both high spin iron(II)), 48,49 in metmyoglobin (high spin iron(III)) 50 and in iron(III)-heme containing HasA, which undergoes a thermal high spin-low spin equilibrium. 34 Proximity of unpaired electron spin(s) to nuclear spins provides them relaxation mechanisms that make nuclear relaxation more efficient.…”