Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)2] in Polar and Protic Solvents.

The [CpFe(CO)(CN)2]- unit is an excellent structural model for the Fe(CO)(CN)2 moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)2] (M1) in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of M1 in H2O, D2O, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN)2 unit. Comparisons of the spectroscopic and dynamic data obtained for M1 in solution and in a dry film with those obtained for the enzyme led to the conclusion that the protein backbone forms an important part of the bimetallic active site environment via secondary coordination sphere interactions.

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir-SI/II, that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.

Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H2, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN- ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail. Using an O2-tolerant [NiFe] hydrogenase as a model system, we demonstrate that IR pump-probe spectroscopy can explore catalytically relevant ligand bonding by accessing high-lying vibrational states. This ultrafast technique also shows that the protein matrix is influential in vibrational relaxation, which may be relevant for energy dissipation from the active site during fast reaction steps. Further insights into the relevance of the active site environment are provided by 2D-IR spectroscopy, which reveals equilibrium dynamics and structural constraints imposed on the H2-accepting intermediate of [NiFe] hydrogenases. Both techniques offer new strategies for uniquely identifying redox-structural states in complex catalytic mixtures via vibrational quantum beats and 2D-IR off-diagonal peaks. Together, these findings considerably expand the scope of IR spectroscopy in hydrogenase research, and new perspectives for the characterization of these enzymes and other (bio-)organometallic targets are presented.

Anterior chamber contamination after uncomplicated phacoemulsification and intraocular lens implantation.

PURPOSE: To determine the frequency of anterior chamber contamination occurring during uncomplicated cataract surgery with intraocular lens implantation using phacoemulsification through a scleral tunnel incision. METHODS: In a prospective study, anterior chamber aspirates of one eye each of 103 consecutive ambulatory patients who underwent uncomplicated cataract surgery with lens implantation were cultured. The anterior chamber aspirate was withdrawn immediately upon the completion of surgery. Conjunctival cultures of the same eye were taken immediately before surgery, after the eye and periocular area had been prepared and draped. Multiple use topical medications used preoperatively on all patients were cultured at the end of the surgical day. RESULTS: Intraocular aspirates yielded positive cultures in five specimens (5%), four of which were identified as Staphylococcus epidermidis. Quantification disclosed colony counts ranging between 100 and 200 colony forming units per milliliter. Results of conjunctival cultures were positive in ten specimens (10%). Staphylococcus epidermidis was the most common isolate, identified in seven of the ten positive cultures. Positive intraocular and conjunctival culture results were not present concurrently in any patient. Microorganisms were recovered from the multiple use topical medications on nine of the 26 successive surgical days. Neisseria species was the most frequent isolate (7,44%), followed by S. epidermidis (5,31%). The frequency of contamination of the anterior chamber was independent of wound width (chi 2 = 0.869) and operative time (chi 2 = 4.77). CONCLUSION: Bacterial contamination of the anterior chamber was detected in five (5%) of the patients. This reduced incidence of contamination compared with that of previous studies may be related to the preoperative preparation, the surgical technique, or both. Contamination of the multiple use topical medications and bulbar conjunctiva define possible sources of pathogens that may enter the anterior chamber. The absence of any clinical ocular infection in all patients attests to the small inoculum sizes, as well as the ability of the anterior chamber to clear small bacterial loads.

Efficacy of topical flurbiprofen and indomethacin in preventing pseudophakic cystoid macular edema. Flurbiprofen-CME Study Group I.

We evaluated two topical cyclo-oxygenase inhibitors (COIs), 0.03% flurbiprofen and 1% indomethacin, for their ability to prevent pseudophakic cystoid macular edema (CME). The study was a randomized, double-masked, vehicle-controlled, parallel group, clinical trial for six months at eight sites in Canada and two in Germany. The study population consisted of 681 patients who had extracapsular cataract extraction and posterior chamber lens implantation. Flurbiprofen, indomethacin, or the vehicle was instilled into the eye four times daily for two days preoperatively and three months postoperatively. Results were measured by angiographic and clinical CME at visit 5 (day 21-60) and visit 7 (day 121-240) and by contrast sensitivity and Snellen visual acuity at all five postoperative visits. At visit 5, the incidence of angiographic CME was comparable in the two COI treatment groups (16.8% flurbiprofen, 12.4% indomethacin) and was significantly lower than in the vehicle group (32.2%). The incidence of clinical CME was also significantly lower in the COI-treated groups (10.7% flurbiprofen, 9.6% indomethacin) than in the vehicle group (21.9%). By visit 7, the incidence of angiographic CME had declined to between 4% and 8% and the incidence of clinical CME was less than 2% in all three groups. At visit 5, contrast sensitivity scores were significantly worse in vehicle-treated patients with angiographic CME than in those without CME, and Snellen visual acuity was one line worse in patients with CME. Flurbiprofen-treated patients achieved good Snellen visual acuity (better than 20/40) sooner than vehicle-treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Risk factors for posterior capsular pearling after uncomplicated extracapsular cataract extraction and plano-convex posterior chamber lens implantation.

We retrospectively reviewed 542 consecutive cases of extracapsular cataract extraction with implantation of a plano-convex laser ridge posterior chamber intraocular lens. We were interested in the incidence and probability of the development of postcapsular epithelial pearling that required laser capsulotomy and the risk factors associated with it. After four years of follow-up, 21% of all cases had developed pearling. Over the same period, the probability of pearling, as determined by the Kaplan-Meier curves, was 29% with a 95% confidence interval between 22% and 36%. After one year of follow-up, the probability of pearling was 5%; after two years, 15%; after three years, 24%. Younger age (50 years or less), larger optic, and smaller IOL were identified as significant risk factors when each of these factors was analyzed separately. When all risk factors were studied together, and after applying confounding statistics, only younger age was identified as a significant risk factor (P = .003).

Argon laser peripheral iridoplasty for angle-closure glaucoma in sibilings with weill-marchesani syndrome.

A patient with Weill-Marchesani syndrome and angle-closure glaucoma had persistent appositional closure after laser iridotomy that was unrelieved by topical application of either miotic or cycloplegic agents. Argon laser peripheral iridoplasty successfully opened the angle. The patient's sister also had Weill-Marchesani syndrome and angle closure unrelieved by laser iridotomy. Angle closure in Weill-Marchesani syndrome and the response to laser iridotomy and treatment with either miotic or cycloplegic agents may be complex and depends on the relative proportion of pupillary block as a mechanism underlying the angle closure, the functional status of the zonular apparatus, and the degree of angle crowding by the peripheral iris in the presence or absence of peripheral anterior synechiae.

Postcataract extraction ptosis: effect of the bridle suture.

Postcataract extraction ptosis is a common complication of cataract surgery. While many factors have been implicated in its etiology, trauma to the superior rectus/levator complex is considered the most important factor. We prospectively evaluated the effect of two superior rectus bridling techniques on the degree and severity of ptosis present three months after cataract surgery. All other potential variables were controlled for. Bridling the tendon of the superior rectus muscle using the direct, subconjunctival (open) approach results in significantly less severe ptosis than the standard technique of indirect transconjunctival (closed) bridling.

Ocular reaction to the use of wet-pack versus dry-pack intraocular lenses.

This study confirms preliminary indications that dry-pack sterilized lenses cause a more marked anterior chamber reaction than wet-pack sterilized lenses during the immediate post-op period. After the first two weeks, this difference is not evident.