James T. Hazzard

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-Å resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170°) and the third (type II) copper lies only 3.1 Å from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 μM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 × 107 M−1⋅s−1), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 × 107 M−1⋅s−1, followed by a slow intramolecular electron transfer to T1 copper (k ∼10 s−1). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

The pH dependence of intramolecular electron transfer rates in sulfite oxidase at high and low anion concentrations

Abstract The individual rate constants for intramolecular electron transfer (IET) between the MoVIFeII and MoVFeIII forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO42– and Cl–, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from FeII to MoVI decreases with increasing pH, both in the presence and absence of 50 mM SO42–. However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO42–, and increases with pH in the presence of 50 mM SO42–. This behavior is consistent with a mechanism in which IET from MoV to FeIII is coupled to proton transfer from MoV–OH to OH–, and the reverse IET process is coupled to proton transfer from H2O to MoVI=O. At high concentrations of small anions, direct access of H2O or OH–to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH– to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase.

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.

Use of laser flash photolysis time-resolved spectrophotometry to investigate interprotein and intraprotein electron transfer mechanisms

A description is given of the methodology developed in our laboratory for the application of laser flash photolysis to the elucidation of the kinetics and mechanism of electron transfer processes which occur intermolecularly between two protein molecules within a collisional complex, or intramolecularly between two redox centers within a single multisubunit or multidomain protein. This involves the use of flavin analogs, excited to their lowest triplet state by a laser flash, to initiate electron transfer, either by oxidation of a sacrificial donor followed by redox protein reduction via the flavin semiquinone, or by direct oxidation of a reduced redox protein by the flavin triplet. Time-resolved spectrophotometry is used to follow the course of the sequence of electron transfer events initiated by the laser flash. The application of this methodology to the following systems is described: cytochrome c/cytochrome c peroxidase; ferredoxin/ferredoxin NADP+ reductase; cytochrome c/plastocyanin; flavocytochrome b2; and sulfite oxidase.

Electron Transfer between the FMN and Heme Domains of Cytochrome P450BM-3 EFFECTS OF SUBSTRATE AND CO

Cytochrome P450BM-3 has the P450 heme domain and FAD/FMN reductase domain linked together in a single polypeptide chain arranged as heme-FMN-FAD. In the accompanying article (Govindaraj, S., and Poulos, T. L. (1997) J. Biol. Chem. 272, 7915-7921, we have described the preparation and characterization of the various domains of cytochrome P450BM-3. One reason for undertaking this study was to provide simpler systems for studying intramolecular electron transfer reactions. In particular, the heme-FMN version of P450BM-3 that is missing the FAD domain should prove useful in studying the FMN-to-heme electron transfer reaction. This version of P450BM-3 has been designated truncated P450BM-3 or BM3t. In this study we have used laser flash photolysis techniques to generate the reduced semiquinone of 5-deazariboflavin which in turn reduces the FMN of BM3t to the semiquinone, FMN, at a rate constant of 6600 s−1, whereas the heme is not reduced by the 5-deazariboflavin radical. The reduction of the heme by FMN does not proceed in the absence of carbon monoxide (CO), whereas in the presence of CO the FMN to heme electron transfer rate constant is 18 s−1. If a fatty acid substrate is present, this rate constant increases to 250 s−1. Somewhat surprisingly, the rate of heme reduction also is dependent on [CO] which indicates that CO causes some change within the heme pocket and/or interaction between the heme and FMN domains that is required for intramolecular electron transfer.

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase

Comparative CO photolysis kinetics studies on wild‐type and autoregulatory (AR) insert‐deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN–heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two‐ to three‐fold, the AR deletion did not change the rate constant for the calmodulin‐controlled IET. This suggests that the rate‐limiting conversion of the electron‐accepting state to a new electron‐donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (FeII) and oxidized molybdenum (MoVI) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k cat. Steady-state kinetic analysis of the Y343F mutant showed an increase inK m sulfite and a decrease ink cat resulting in a 23-fold attenuation in the specificity constantk cat/K m sulfiteat the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.

Binding of YC-1 or BAY 41-2272 to Soluble Guanylyl Cyclase Induces a Geminate Phase in CO Photolysis

Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of approximately 150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 x 10(7) s(-1). These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.

Direct measurement by laser flash photolysis of intramolecular electron transfer in the three-electron reduced form of ascorbate oxidase from zucchini

Ascorbate oxidase, which has been fully reduced by its substrate, can rapidly transfer a single electron to the laser-generated triplet state of 5-deazariboflavin. Subsequent to this, intramolecular electron transfer occurs resulting in the oxidation of the blue type I copper center. This latter process proceeds via biphasic kinetics, with observed rate constants of 9500 s-1 and 1400 s-1, both of which are protein concentration independent. This indicates that the initial oxidation reaction involves the type II, III trinuclear center, probably occurring via parallel reactions of two of the three copper atoms. The rate constants for intramolecular electron transfer in the three-electron reduced enzyme are one to two orders of magnitude larger than previously observed for the one-electron reduced enzyme, indicating a dramatic effect of the redox state of the enzyme on the intramolecular communication between the copper centers.

Proton NMR study of the cytochrome c: Flavodoxin electron transfer complex

The effects of complex formation with flavodoxin on the proton NMR spectrum of cytochrome c are to change the resonance frequencies and to increase the bandwidths of most of the low and high field heme, Met-80, and His-18 protons. These effects are, in general, more pronounced than has been reported for other cytochrome c complexes. The degree of line broadening for many heme related resonances suggests that complex formation induces changes in the cytochrome structure. These results provide the first spectroscopic evidence which corroborates the proposed model for the cytochrome c: flavodoxin complex (1-3).