Colin Greenwood

Magnetic Circular Dichroism of Hemoproteins.

Publisher Summary This chapter describes the progress made over the last 10 years in studies of the magnetic circular dichroism (MCD) spectra of heme models and proteins. The construction by Oxford Instruments of a split-coil superconducting magnet, in which the sample could be immersed in a liquid helium bath, necessitated the development of optical windows that remained strain free and capable of propagating circularly polarized light at 4.2 K. This led to the ability to make measurements routinely on samples of hemoproteins over the temperature range 1.5–300 K and up to fields of 6–7 T and opened the way for the measurement for the first time of MCD magnetization curves of metalloproteins and hence the methodology for the study of the ground-state magnetic properties of individual heme centers in proteins. It is now possible to measure the MCD spectra of hemoproteins in a solution of water and cryoprotectant mixtures over the wavelength range 195–5000 nm at magnetic fields up to 10 T, with good sample temperature control over the range 1.5–300 K. Although MCD studies in the vacuum ultraviolet region have been carried out using laboratory-based instruments and synchrotron sources, no reports have appeared of the spectra of hemoproteins in this wavelength region.

Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa

BACKGROUND Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (-330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin. RESULTS The crystal structure of the oxidized form of PsCCP has been determined to 2.4 A resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands. CONCLUSIONS The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce a new residues into the haem pocket.

The Dinuclear Center of Cytochrome bo3 from Escherichia coli

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively charactererized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

Studies of cyanide binding to myeloperoxidase by electron paramagnetic resonance and magnetic circular dichroism spectroscopies

Abstract The techniques of magnetic circular dichroism (MCD) and absorbance spectroscopy have been used to monitor titrations of oxidized myeloperoxidase with CN − , at room temperature, over the Soret, visible and near-infrared regions of the spectrum. Electron paramagnetic resonance measurements have also been made between 10–30 K. The near-infrared MCD of the unbound enzyme possesses a bi-signate band at 1000 nm, similar to those seen with other high-spin haemoproteins. On addition of CN − this band is replaced by a positive band at 1500 nm, which is assigned to low-spin haem. This switch in spin state was also observed by EPR spectroscopy with high-spin signals at g 7.09 and 5.17 titrating away to low-spin signals at g 2.58, 2.33 and 1.81. Significant changes were apparent even at ratios of CN − /haem of less than one. Integrations of the EPR spectra bave been made to check absorbance spectrum extinction coefficients, in the absorbance and MCD spectra measured over the Soret and visible bands evidence was found for more than one CN − binding process. The MCD spectrum of cyanide-bound oxidized myeloperoxidase from 350 to 700 nm strongly suggests that the haem is closely related to a ring of the chlorin type.

The structure of the cytochrome a3-CuB site of mammalian cytochrome c oxidase as probed by MCD and EPR spectroscopy.

The nature of the complexes formed between cytochrome c oxidase and the three inhibitory ligands N3-, CN-, and S2- have been investigated by a combination of MCD and EPR spectroscopy. CN- forms a linear bridge between the Fe III a3 and CuB II, suggesting that the distance between these centers in the oxidized enzyme is between 5 and 5.25 A. This distance is too short to permit N3- to form a linear bridge and the evidence suggests this to be bent. In contrast S2- or SH- is unable to form any bridge and it seems likely that two SH- ions are bound by the bimetallic site, one to Fe III a3 and the other to CuB I. The significance of the a3-CuB distance in terms of oxygen binding and reduction is discussed.

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow‐flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t =5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.

Determination of the optical properties of CuA(II) in Bovine Cytochrome c oxidase using magnetic circular dichroism as an optical detector of p

This paper describes the use of a novel magnetic circular dichroism-microwave double resonance (MCD-ODMR) experiment to study the optical properties of the EPR detectable copper center, CuA2+, in bovine cytochrome c oxidase. By irradiating the sample with a monochromatic microwave beam of appropriate frequency it is possible to quench partially the MCD intensity of the features due to CuA2+. In this way the MCD bands from this center have been identified even in the presence of overlapping optical transitions from the haem centers of this enzyme. The resulting spectrum compares well with that reported some years ago from this laboratory and obtained by measuring MCD magnetisation characteristics. In addition the shapes of the MCD-ODMR lines obtained in a plot of MCD intensity against magnetic field strength have been analyzed to yield the relative polarizations of the optical transitions of CuA2+ which contribute to the MCD spectrum. All of the bands observed between 450 and 850 nm are predominantly polarized in the xy plane perpendicular to the direction of the g-tensor component of CuA2+ at g parallel = 2.18. This suggests that all of the CuA2+ ligands that contribute to the optical charge-transfer transitions in the visible region lie approximately in the basal plane. Possible structures for CuA2+ can now be suggested.

Electron paramagnetic resonance and magnetic circular dichroism studies of a hexa-heme nitrite reductase from Wolinella succinogenes.

The nature of the heme centers in the hexa‐heme dissimilatory nitrite reductase from the bacterium Wolinella succinogenes has been investigated with EPR and magnetic circular dichroism spectroscopy. The EPR spectrum of the ferric enzyme is complex showing, in addition to magnetically isolated low‐spin ferric hemes with g values of 2.93, 2.3 and 1.48, two sets of signals at g = 10.3, 3.7 and 4.8, 3.21, which we assign to two pairs of exchange coupled hemes. The MCD spectra show that the isolated hemes are bis‐histidine coordinated and that there is one high‐spin ferric heme. The exchange coupling is lost on treatment with SDS.

Distinct forms of the haem o-Cu binuclear site of oxidised cytochrome bo from Escherichia coli: Evidence from optical and EPR spectroscopy

Oxidised, formate‐bound and fluoride‐bound forms of E. coli cytochrome bo give rise to an electronic absorption band near 630 nm, diagnostic of high‐spin ferrric haem o, whose position is sensitive to the nature of the bound anion. In all three forms, haem o remains spin‐coupled to cuB(II), resulting in distinct broad X‐band EPR signals. Those of formate‐bound cytochrome bo are similar to the signals seen in slow cytochrome aa 3 but cannot be induced by incubation at acid pH suggesting that the endogenous earboxylate believed to be important in slow cytochrome aa 3 is not present in cytochrome bo. The oxidised form gives rise to novel EPR signals at g = 3.74 and g = 3.08 which have not been detected in cytochrome aa 3 and may arise from a weak magnetic coupling between high‐spin haem o, S = 5/2, and Cub(ii), S = ½.

The reaction of Escherichia coli cytochrome bo with H202: Evidence for the formation of an oxyferryl species by two distinct routes

We have re‐examined the reaction of fast oxidised cytochrome bo with H2O2 in a stopped‐flow spectrophotometer. Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469–475 [1]). The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence. Initially these results appeared incompatible with the report that only one equivalent of H2O2 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) Eur. J. Biochem. 226, 731–737 [2]). However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.