Célia V. Romão

The nature of the di-iron site in the bacterioferritin from Desulfovibrio desulfuricans

The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 Å resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.

Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

Abstract A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c3. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c3, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c3. A similar study with D. vulgaris Hildenborough cytochrome c553 shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c3 have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases.

The Role of the Hybrid Cluster Protein in Oxidative Stress Defense

Hybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S]2+/1+ or [2Fe-2S]2+/1+ cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited Km values for hydrogen peroxide (∼0.3 mm) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection.

The crystal structure of Deinococcus radiodurans Dps protein (DR2263) reveals the presence of a novel metal centre in the N terminus

The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-Å resolution, and one soaked in an iron solution, to 1.6-Å resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage.

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans.

A bacterioferritin was recently isolated from the anaerobic sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841–6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron‐coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate‐reducing bacteria.

The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism.

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di‐iron centre and having iron‐coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin‐2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin‐2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin‐2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin‐2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin‐2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.

Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris hildenborough.

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.

Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin.

Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7°C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100mM MES pH 6.2, 500mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.

Flavodiiron Oxygen Reductase from Entamoeba histolytica MODULATION OF SUBSTRATE PREFERENCE BY TYROSINE 271 AND LYSINE 53

Background: Flavodiiron proteins (FDPs) are O2 and/or NO reductases. Results: Point mutations, near the active site of an Entamoeba histolytica O2-selective FDP, result in increased NO reductase activity and faster inactivation in the reaction with O2. Conclusion: Residues close to the FDPs diiron site modulate the preference toward O2 or NO. Significance: We unravel molecular determinants of substrate specificity in enzymes affording resistance to oxygen and/or nitric oxide. Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity.

The dual function of flavodiiron proteins: oxygen and/or nitric oxide reductases

Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.