Nikolaos Ioannidis

Reactions of the Escherichia coli Flavohaemoglobin (Hmp) with Oxygen and Reduced Nicotinamide Adenine Dinucleotide: Evidence for Oxygen Switching of Flavin Oxidoreduction and a Mechanism for Oxygen Sensing

The soluble flavohaemoglobin (Hmp) of Escherichia coli contains haem B and FAD in a single 44 kDa polypeptide, and shows NADH oxidase activity. The oxidized protein reacted rapidly with NADH in the presence of O2 to form an oxygenated species while the flavin remained largely oxidized. Spectral and kinetic analyses revealed rapid biphasic reduction and oxygenation of high-spin haem with apparent relaxation times of 6 and 64 ms at pH 8 and 25 °c, suggestive of a significant physiological role for the protein. This was followed by a monophasic reduction of the flavin with a relaxation time of 92 ms. On exhaustion of oxygen, the oxygenated haem was converted into the deoxy form biphasically with relaxation times of 43 and 170 s, followed by extensive reduction of the flavin with corresponding relaxation times of 70 and 256 s. Based on these observations, we propose that Hmp could act as an oxygen sensor in E. coli by combining with intracellular oxygen, thus limiting flavin reduction in the aerobic steady state. Lowering of the oxygen concentration causes dissociation of the oxy species and sustained flavin reduction. Because Hmp can reduce Fe(III), such a mechanism might control, for example, flavin-mediated Fe(III) reduction required for activation of the anaerobic gene regulator, Fnr.

The oxygenated flavohaemoglobin from Escherichia coli: evidence from photodissociation and rapid-scan studies for two kinetic and spectral forms.

The kinetics of dissociation and reassociation of the oxygenated species of Escherichia coli flavohaemoglobin (Hmp) were studied using stopped-flow rapid-scan and flash photolysis spectrophotometry at 25 degrees C. The oxygenated compound(s) form rapidly on mixing oxygen with the NADH-reduced flavohaemoglobin. On exhaustion of NADH, with residual oxygen, decay occurs in two phases to give a form in which haem b and flavin are oxidized. Spectral changes during this process suggest a direct release of O2- from the oxy form. Photodissociation of the oxygenated species generates the unliganded protein, which recombines with oxygen to give two spectrally and kinetically distinct forms. The reversibility of the oxygen reaction and the rapid reassociation kinetics after photodissociation confirm the haemoglobin-like features of this protein.

The EPR Spectrum of Tyrosine Z• and Its Decay Kinetics in O2-Evolving Photosystem II Preparations†

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.

Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

We compare the interaction of nitric oxide with the S states of the oxygen evolving complex (OEC) of photosystem II and the dinuclear Mn cluster of Thermus thermophilus catalase. Flash fluorescence studies indicate that the S3 state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S1 with an apparent halftime of ca. 0.4 s at about 18°C, compared with a biphasic decay, with approximate halftimes of 28 s for S3 to S2 and 140 s for S2 to S1 in the absence of NO. Under similar conditions the S2 state is reduced by NO to the S1 state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S1 state at − 30°C, via the S0 and S−1 states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S3 state of the OEC, which is compatible with a metalloradical character of this state.

Conversion of the g = 4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S(0)-S(1),...,S(3)-(S(4))S(0) during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)(4)Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S(2) shows a notable heterogeneity with two spectral forms; a g=2 (S=1/2) multiline, and a g=4.1 (S=5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S(3) they exhibit the same characteristic g=10 (S=3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g=4.1 conformation during advancement to S(3) is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S(2) to S(3) transition (ca 230K). We observed that, above about 220K the g=4.1 signal converts to a multiline form prior to advancement to S(3). We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S(2) to S(3) transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)(4)Ca with a low-spin ground state advances to S(3).

Can we trap the metalloradical intermediate during the S‐state transitions of Photosystem II? An EPR investigation

We report the trapping of two metalloradical intermediates corresponding to the transitions S2 to S3 and S3 to S0 of the oxygen evolving complex (OEC) of Photosystem II (PSII), in preparations containing methanol, at temperatures near that of half inhibition of the respective S‐state transitions. The first intermediate, with an EPR width of 160 G, is assigned to , based on its similarity to the one previously characterized after trapping at 10 K. The second with a splitting of ∼80 G is tentatively assigned to . The EPR signal is weaker than the one, and both are stable at cryogenic temperatures.

The Progressive Exchange-Narrowing of the S0YZ •, S1YZ •, and S2YZ • Spectra Reveals the Unperturbed Spectrum of Tyr Z• in Oxygen Evolving PSII Preparations: A Rapid Scanning EPR Investigation in the Temperature Range 4.2–240 K

We studied the temperature dependence of the S1YZ •, S2YZ •, S0YZ •(+MeOH) and S2YZ •(+MeOH) metalloradical EPR signals in the temperature range 4.2–240 K, using slow and rapid scans. As the temperature increases the spectra narrow progressively and above 100 K collapse to a “25 G” signal somewhat broader than “signal II”. The spectra do not reach saturation at microwave powers up to 100 mW over the entire temperature range, and the signal intensity multiplied by temperature remains approximately constant. The narrowing of the SnYz • EPR signals is consisted with an increase of the Mn spin-lattice relaxation rate, with increasing temperature. We conclude that the broad EPR signals observed at low temperatures and the narrow signals at elevated temperatures are manifestations of the same intermediate SnYz • (n = 0, 1, 2) and the high temperature spectrum is due to Tyr Z• unperturbed by the magnetic interaction with Mn.

Probing Tyrosine Z• of the Functional Photosystem II at Temperatures Close to the Onset of the S-state Transitions: An EPR Investigation Employing Rapid Scans

The Magnetically-Split Spectra Of The Metalloradical Intermediates S0YZ • And S2,Yz • Collapse Above ∼100 K To The Unperturbed Spectrum Of YZ • (Zahariou Et Al. 2007). We Studied The Spectrum Of YZ •As Well As Its Decay Kinetics Above 100 K In Various S States. In This Report The Intermediate S2YZ •Is Examined. Its Unperturbed Spectrum Appears Distinct From That Of The Stable YD • Radical. The Spectrum Of YZ • Was Simulated By Varying The Rotational Conformation Of The Phenoxyl Ring And The Spin Density p On Carbon C1. The Latter Can Be Related To The Strength Of The Hydrogen Bond Between Tyrosine And Its Base Partner (Presumably D1 His190).

Intermediates of the 3 state of the oxygen evolving complex of photosystem II. (1) Decay products

Near-IR light excitation of the S3 state produces, in addition to other signals that appear to be associated with excited state configurations of S3, a derivative-shaped EPR signal at g ~ 5.1,2 This signal bears unexpected similarities to the one observed earlier by Nugent et al.3, in samples that had undergone multiple turnovers above S1 and subsequently stored at 77 K for a week or longer. We have confirmed the observations of Nugent et al.3 and furthermore shown that, the g ~ 5 signal evolves in apparent correlation with the decay of the S3 state. The g ~ 5 signal that evolves slowly at 77 K is super-imposable and shares similar EPR properties with the one induced by NIR excitation of S3. At temperatures > -80 0C the S2 multiline and g = 4.1 signals grow in parallel during the decay of the g ~ 5 or the freshly prepared S3. It is suggested that, the broad g ~ 2 radical that forms immediately after excitation of the S3 state by NIR light2 arises from centers where the positive hole was transferred from the Mn cluster to YZ. These centers rapidly recombine to yield an excited S2 state configuration identical to the one that is produced during the slow decay of S3 at 77 K. Theoretical simulations of the g ~ 5 signal will be presented.