Femtosecond X-ray spectroscopy of haem proteins

We discuss our recently reported femtosecond (fs) X-ray emission spectroscopy results on the ligand dissociation and recombination in nitrosylmyoglobin (MbNO) in the context of previous studies on ferrous haem proteins.

On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.

Spectroscopic analysis of protein Fe–NO complexes

The toxic free radical NO (nitric oxide) has diverse biological roles in eukaryotes and bacteria, being involved in signalling, vasodilation, blood clotting and immunity, and as an intermediate in microbial denitrification. The predominant biological mechanism of detecting NO is through the formation of iron nitrosyl complexes, although this is a deleterious process for other iron-containing enzymes. We have previously applied techniques such as UV–visible and EPR spectroscopy to the analysis of protein Fe–NO complex formation in order to study how NO controls the activity of the bacterial transcriptional regulators NorR and NsrR. These studies have analysed NO-dependent biological activity both in vitro and in vivo using diverse biochemical, molecular and spectroscopic methods. Recently, we have applied ultrafast 2D-IR (two-dimensional IR) spectroscopy to the analysis of NO–protein interactions using Mb (myoglobin) and Cc (cytochrome c) as model haem proteins. The ultrafast fluctuations of Cc and Mb show marked differences, indicating altered flexibility of the haem pockets. We have extended this analysis to bacterial catalase enzymes that are known to play a role in the nitrosative stress response by detoxifying peroxynitrite. The first 2D-IR analysis of haem nitrosylation and perspectives for the future are discussed.

Relationship between protein structural fluctuations and rebinding dynamics in ferric haem nitrosyls

The interaction of nitric oxide (NO) with haem proteins is widespread in biology. In the current paper, we present the first ultrafast 2D-IR (two-dimensional infrared) spectroscopic analysis of haem nitrosylation, which has been combined with time-resolved IR pump–probe studies to investigate the relationship between equilibrium vibrational dynamics of the haem environment and ligand rebinding behaviour following photolysis of NO from the Fe(III)–NO site. Studies of two haem proteins, Mb (myoglobin) and Cc (cytochrome c), which play different physiological roles, reveal marked contrasts in the ultrafast fluctuations of the protein pockets containing the haem, showing that the Mb pocket is somewhat more flexible than that of Cc. This correlates strongly with slower observed photolysis rebinding kinetics of Mb–NO compared with Cc–NO, and indicates a direct link between ultrafast fluctuations and biological functionality. Furthermore, this indicates the validity of linear response theories in relation to protein ligand binding. Finally, 2D-IR shows that Cc–NO displays two distinct structural sub-sites at room temperature that do not exchange on the timescales accessible via the NO vibrational lifetime.

Iron metabolism and its disorders

Iron is a component of haem proteins and nonhaem enzyme systems required for oxygen transport, mitochondrial respiration, and other key metabolic reactions. The metal exists in two readily interconvertible redox states (divalent ferrous and trivalent ferric iron) that are highly reactive and toxic to tissues. High-affinity iron-binding proteins, which form stable ferric complexes, have evolved to facilitate iron transport and delivery to sites of storage and utilization, including haem biosynthesis....