Resolution of two distinct electron transfer sites on azurin

Farver, Ole; Blatt, Yoav; Pecht, Israel

doi:10.1021/bi00258a005

Cited by 45 publications

(32 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3A), from which a 3.0 s-' exchange rate is calculated using a T, = 83 ms for the high-pH signal J. This value is consistent with the rate constant for the acid-base exchange process of the native copper-azurin estimated by means of kinetic methods [55,561.…”

Section: Resultssupporting

confidence: 82%

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

Salgado

Jiménez

Donaire

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 'H-NMR spectrum of Pseudornonas aeruginosa Co(I1)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(I1)-azurin, i.e. His46, Hisll7, Cysll2, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(I1) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N,S plane towards the Gly45 0 axial position and weakly interacting with the Met121 sulfur.Keywords. Azurin; paramagnetic NMR; metal substitution; type 1 copper proteins ; cobalt proteins.Substitution of the naturally occumng metals in metalloproteins is used as a common strategy in inorganic biochemistry [l, 21. In a first approach it can be considered as a kind of sitedirected mutation, the most simple we can think of in the case of a metalloprotein. In this sense, apart from the possible function of the metal, it can give an idea of the metal site selectivity and structural rigidity, the mechanism of metal uptake, some general chemical properties of the metalloprotein, etc. In some special cases metal substitution is used to probe the metal site [ 1 -31. This is possible when the chromophore that brings along the exogenous metal has peculiar spectroscopic properties. For the blue copper proteins, like azurin, the natural copper(I1) center has itself singular, informative and in some way exciting spectroscopic properties (41. These are principally EPR and electronic spectral characteristics whose extensive study throughout the last few decades has achieved a detailed knowledge of type 1 copper sites [4-61. But even in this case metal substitution has proved to be useful in solving fundamental questions such as the assignment of the main visible bands of the blue copper proteins as S(Cys)-metal charge transfer bands Abbreviations. COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy ; WEFT, water-eliminated Fo...

show abstract

Section: Resultssupporting

confidence: 82%

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

Salgado

Jiménez

Donaire

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Previous studies (32)(33)(34)(35)(36)(37)(38) used the same concept to qualitatively establish the binding site of chromium(III) in oxidation reactions involving chromium(II) and assuming that the binding site of chromium(II) is the same as that of chromium(III). However, in such systems the binding of chromium was found often to occur at multiple sites.…”

Section: Concluding Remarks and Implications For Cyt Cmentioning

confidence: 99%

“…It is kinetically inert as far as ligand exchange is concerned (16). For all these reasons, it can be used as an efficient relaxation reagent, and indeed it has been used as such to probe specific binding sites in many systems (32)(33)(34)(35)(36)(37)(38). However, the present study represents an attempt to exploit these effects to calculate the structure of the metal-protein adduct.…”

mentioning

confidence: 99%

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

Assfalg

Bertini

Bruschi

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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...

show abstract

“…Azurin is a blue single-copper protein that functions as an electron carrier physiologically associated with oxidative stress responses in bacteria (e.g., Pseudomonas aeruginosa) (26) and is a long-standing model for exploring electron tunneling through protein molecules (27)(28)(29)(30)(31)(32). Thanks to its intrinsic merits (e.g., high stability and excellent redox properties), azurin has recently emerged as a favorite target for nanoscale bioelectronics (33,34).…”

mentioning

confidence: 99%

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

Chi

Farver

Ulstrup

2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

173

222

View full text Add to dashboard Cite

A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 Å ؊1 in H2O and D2O, respectively. Redoxgated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on͞off current ratio of Ϸ9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.blue copper protein ͉ scanning tunneling microscopy ͉ nanoscale bioelectronics ͉ bioelectrochemistry C harge transfer plays key roles in many chemical and biological processes as well as in molecular electronics (1-5). For example, long-range protein electron transfer (ET) is central in aerobic respiration and photosynthesis. The importance of longrange ET is illustrated by a recent special issue of PNAS on this topic (6-12). Several articles reflect broadly the current status of this subject. However, one of the major objectives in nanoscale science and technology is to fabricate molecular electronic devices with specified functions. Molecular electronics is rooted in the concept of molecular charge transfer (particularly, molecular conductivity) (13,14). Two essential steps involved in bottom-up manipulations are (i) organizing molecules into nanoscale structures and (ii) interfacing such nanostructures with macroscopically addressable components (e.g., metal and semiconductor electrodes). Molecular electronic device function thus rests fundamentally on charge transfer through organic and͞or biological molecules and across the interface between molecules and macroscopic electrodes (15, 16). Understanding of charge transfer mechanisms has mostly been based on average results of macroscopic measurements. The advent of scanning probe microscopies, along with other supersensitive techniques, has made it possible to characterize or directly observe charge transfer through organic molecules down to the nanoscale and single-molecule levels, as illustrated by measurements of singlemolecule conductivity (17-21) and probing of molecular switching and resonant tunneling (22,23). This, however, remains a daunting challenge for proteins, with difficulties arising from the assembly of suitable st...

show abstract

Resolution of two distinct electron transfer sites on azurin

Cited by 45 publications

References 27 publications

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

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

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

Contact Info

Product

Resources

About

Resolution of two distinct electron transfer sites on azurin

Cited by 45 publications

References 27 publications

1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

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

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

Contact Info

Product

Resources

About

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

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