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

Abstract

The redox reaction between CrO 4 2Ϫ and the fully reduced threeheme cytochrome c 7 from Desulfuromonas acetoxidans to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 ؎ 0. S ulfur-and sulfate-reducing bacteria constitute a group of anaerobic organisms, the common feature of which is the use of sulfur and its oxidized forms as electron acceptors. Thiosulfate, sulfur, sulfite, or sulfate ''respiration'' produces sulfide. The end product of the reaction, hydrogen sulfide, can react with heavy metal ions to form less toxic insoluble metal sulfides (1). These bacteria are also able to enzymatically reduce and precipitate heavy metals (2-8). They therefore are important for the geochemical cycle of metals and become particularly relevant for possible applications in the decontamination of environments polluted by toxic heavy metals through bioreduction coupled with precipitation as insoluble sulfides. Among the metals that can be reduced by these bacteria, chromium(VI) is particularly relevant from the technological and environmental point of view, because it represents one of the most common polluting metals and is highly soluble and toxic. § 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. 11 and 15) prompted us to study by NMR the reaction between CrO 4 2Ϫ and fully reduced Cyt c 7 . The products of the reaction are chromium(III) and the oxidized protein with three iron(III) hemes. Because chromium(III) is kinetically inert with respect to ligand exchange, it remains bound to protein residues through which electron transfer occurs. The hyperfine coupling between the unpaired electrons of chromium(III) and the proton nuclei of the protein gives rise to particular features in the NMR spectra (16) that could be used to obtain structural information about the chromium(III) binding site. The redox-inert MoO 4 2Ϫ was used to map the binding site of the anion. NMR has allowed us to obtain a clear picture of the...

Metabolism of Metals and Metalloids by the Sulfate-Reducing Bacteria

“…This process is interesting because it is proposed that hydrogen sulfide produced by the sulfate reducing bacteria destabilizes the Au(S 2 O 3 ) 2 − complex resulting in the reduction of Peaks indicating presence of arsenic are highlighted in red, iron in blue and sulfur in orange. Inset (B) is a selected-area electron diffraction (SAED) pattern indicating crystalline order of arsenic deposits.…”

“…The cytochrome c 7 from Desulfuromonas acetoxidans has provided interesting information about chromium reduction [2]. Cytochrome c 7 has three heme groups and Cr(VI) binds onto the protein at a site that is closer to heme IV than to heme III or heme I.…”

“…This process involves substrate-or non-substrate specific enzymatic enzymes that are respiratory components of the cells. While cytochrome c 3 has been demonstrated to reduce specific metallic elements, enzymes and electron carriers with metal cofactors may also have the capability to reduce metal(loid)s. The cytochromes from sulfate/sulfur reducers are unique and it has been suggested by Lojou et al [55], Assfalg et al [2] that cytochromes (i.e., c 3 and c 7 ), with the bishistidinyl heme iron coordination, act as metal reducers. The interaction of a detoxifying protein with many different metals has potential practical applications.…”

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

“…However, microbial, enzymatic, and chemical reduction of Cr(VI) has been shown to produce significant amounts of soluble Cr(III), e.g., (Shen and Wang 1993;Campos et al 1995;Buerge and Hug 1998;McLean and Beveridge 2001;Assfalg et al 2002;Puzon et al 2002Puzon et al , 2005Bencheikh-Latmani et al 2007). The soluble Cr(III) forms are likely organoCr(III) complexes.…”

“…First, Cr(VI) reduction by several bacterial species has resulted in the production of soluble Cr(III) in the supernatant (Shen and Wang 1993;Campos et al 1995;McLean and Beveridge 2001;Megharaj et al 2003;BencheikhLatmani et al 2007). Second, enzymatic reductions of Cr(VI) have formed organo-Cr(III) complexes, where Cr(III) is bound to the oxidized form of the reductants, such as NAD + (Puzon et al 2002;Kwak et al 2003) and cytochrome c 7 (Assfalg et al 2002). Third, reduction of Cr(VI) in the presence of organics and common intracellular components produces various soluble organo-Cr(III) complexes (Buerge and Hug 1998;Puzon et al 2005).…”