PDBsum entry 3in2

Electron transport

PDB id: 3in2

Contents

Protein chain

128 a.a. *

Metals

_CU

Waters ×84

* Residue conservation analysis

PDB id:

3in2

Name:

Electron transport

Title:

Crystal structure of the n47s/m121l variant of pseudomonas aeruginosa azurin in the cu(ii) state

Structure:

Azurin. Chain: a. Engineered: yes. Mutation: yes. Other_details: n47s/m121l variant of azurin

Source:

Pseudomonas aeruginosa. Organism_taxid: 287. Gene: azu, pa4922. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.60Å R-factor: 0.238 R-free: 0.266

Authors:

Y.G.Gao,H.Robinson

Key ref:

N.M.Marshall et al. (2009). Rationally tuning the reduction potential of a single cupredoxin beyond the natural range. Nature, 462, 113-116. PubMed id: 19890331 DOI: 10.1038/nature08551

Date:

11-Aug-09 Release date: 17-Nov-09

Protein chain

P00282  (AZUR_PSEAE) -  Azurin from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 148 a.a.

Struc: 128 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1038/nature08551

Nature 462:113-116 (2009)

PubMed id: 19890331

Rationally tuning the reduction potential of a single cupredoxin beyond the natural range.

N.M.Marshall, D.K.Garner, T.D.Wilson, Y.G.Gao, H.Robinson, M.J.Nilges, Y.Lu.

ABSTRACT

Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. These functions are accomplished in nature by only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centres or fuel cell catalysts for energy conversion. Here we show that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials.

Selected figure(s)

Figure 1.
Figure 1: X-ray structures of Az and selected variants. a, Native azurin (PDB 4AZU). b, N47S/M121L azurin: N47S affects the rigidity of the copper binding site and, probably, the direct hydrogen bonds between the protein backbone and Cys 112. c, N47S/F114N azurin: introducing a hydrogen-bond donor at position 114 perturbs hydrogen-bonding near the copper binding site, possibly disrupting donor–acceptor interactions to His 117, or ionic interactions between the copper and the carbonyl oxygen of Gly 45. d, F114P/M121Q azurin: F114P deletes a direct hydrogen bond to Cys 112 resulting in a lower redox potential. The ultraviolet–visible spectroscopy of the F114P-containing variants shows a significant increase in the copper d arrow d absorbance range around 800 nm. This increased absorbance suggests slight rearrangement of the copper binding site, but is consistent with F114P Az^27 and other T1 copper proteins, such as plastocyanin^9. In all panels copper is shown in green, carbon in cyan, nitrogen in blue, oxygen in red and sulphur in yellow. Hydrogen-bonding interactions are shown by dashed red lines.

Figure 2.
Figure 2: Rational tuning of the reduction potential of Az. a, Plot of E^o at pH 7.0 versus log P for the Az mutants from this study (here P is the partition coefficient of the side chain of the residue between octanol and water; that is, it is a measure of hydrophobicity). The lowest reported E^o (dashed orange line) at pH 7.0 for any T1 cupredoxin before this study, M86Q pseudoazurin, is indicated. The highest E^o (dashed purple line) at pH 6.2 of any T1 cupredoxin variant before this study, M148L Rc, is also indicated; its potential was not measured at pH 7.0 owing to protein instability. Considering the pH trend of the E^o of all cupredoxins, this reported value for M148L Rc would be lower at pH 7.0. b, Plot showing the E^o value for each azurin variant at pH 7.0 unless otherwise noted. Not only is the redox potential of Az tunable to the extremes of the redox potentials attainable by T1 cupredoxins, but to nearly any redox potential within the range. Error bars, 1 s.d.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2009, 462, 113-116) copyright 2009.