A trade-off between plant and soil carbon storage under elevated CO2.

Terrestrial ecosystems remove about 30 per cent of the carbon dioxide (CO2) emitted by human activities each year1, yet the persistence of this carbon sink depends partly on how plant biomass and soil organic carbon (SOC) stocks respond to future increases in atmospheric CO2 (refs. 2,3). Although plant biomass often increases in elevated CO2 (eCO2) experiments4-6, SOC has been observed to increase, remain unchanged or even decline7. The mechanisms that drive this variation across experiments remain poorly understood, creating uncertainty in climate projections8,9. Here we synthesized data from 108 eCO2 experiments and found that the effect of eCO2 on SOC stocks is best explained by a negative relationship with plant biomass: when plant biomass is strongly stimulated by eCO2, SOC storage declines; conversely, when biomass is weakly stimulated, SOC storage increases. This trade-off appears to be related to plant nutrient acquisition, in which plants increase their biomass by mining the soil for nutrients, which decreases SOC storage. We found that, overall, SOC stocks increase with eCO2 in grasslands (8 ± 2 per cent) but not in forests (0 ± 2 per cent), even though plant biomass in grasslands increase less (9 ± 3 per cent) than in forests (23 ± 2 per cent). Ecosystem models do not reproduce this trade-off, which implies that projections of SOC may need to be revised.

Experimental testing of hypotheses for temperature- and pH-based niche specialization of ammonia oxidizing archaea and bacteria.

Investigation of niche specialization in microbial communities is important in assessing consequences of environmental change for ecosystem processes. Ammonia oxidizing bacteria (AOB) and archaea (AOA) present a convenient model for studying niche specialization. They coexist in most soils and effects of soil characteristics on their relative abundances have been studied extensively. This study integrated published information on the influence of temperature and pH on AOB and AOA into several hypotheses, generating predictions that were tested in soil microcosms. The influence of perturbations in temperature was determined in pH 4.5, 6 and 7.5 soils and perturbations in pH were investigated at 15°C, 25°C and 35°C. AO activities were determined by analysing changes in amoA gene and transcript abundances, stable isotope probing and nitrate production. Experimental data supported major predictions of the effects of temperature and pH, but with several significant discrepancies, some of which may have resulted from experimental limitations. The study also provided evidence for unpredicted activity of AOB in pH 4.5 soil. Other discrepancies highlighted important deficiencies in current knowledge, particularly lack of consideration of niche overlap and the need to consider combinations of factors when assessing the influence of environmental change on microbial communities and their activities.

Underlying lithology controls trace metal mobilization during redox fluctuations.

Redox state fluctuations are a primary mechanism controlling the mobilization of trace metals in soils. However, underlying lithology may modulate the effect that redox fluctuations have on trace metal mobility by influencing soil particle size and mineral composition. To investigate the relationships among trace metal behavior, lithology, and redox state, we subjected surface soils from two intensely weathered soil profiles formed on contrasting lithologies to consecutive, 8-day redox cycles. A suite of metals (Al, Mn, Fe, Ti, Rb, Zr, Nb, Mo, REEs, Pb, Th, U) were quantified in the aqueous phase (<10 nm) and solution (<415 nm, including colloids) from soil slurries. In soil formed on volcaniclastic bedrock with high clay content and a high abundance of short-range-ordered Fe-(oxyhydr)oxides phases (e.g. nano-goethite; quantified by Mössbauer spectroscopy), reducing events and colloidal dynamics drove trace metal mobilization. In contrast, in soil formed on granite bedrock with lower clay content and a low abundance of short-range-ordered Fe-(oxyhydr)oxides phases (nano-goethite and lepidocrocite), overall trace metal mobilization was lower, and mobilization was not predictable from redox state. Molybdenum isotopes were also measured through redox cycles but did not exhibit redox-dependent behavior. This study provides direct evidence that lithology remains an overarching factor governing the characteristics of metal mobility in soils, even after extended and intense chemical weathering and soil development processes.

AHNS Series: Do you know your guidelines? AHNS Endocrine Section Consensus Statement: State-of-the-art thyroid surgical recommendations in the era of noninvasive follicular thyroid neoplasm with papillary-like nuclear features.

The newly introduced pathologic diagnosis of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) will result in less bilateral thyroid surgery as well as deescalation in T4 suppressive and radioactive iodine treatment. Although, NIFTP is a nonmalignant lesion that has nuclear features of some papillary malignancies, the challenge for the surgeon is to identify a lesion as possibly NIFTP before the pathologic diagnosis. NIFTP, due to its reduction of overall rates of malignancy, will result in the initial surgical pendulum swinging toward lobectomy instead of initial total thyroidectomy. This American Head and Neck Society endocrine section consensus statement is intended to inform preoperative evaluation to attempt to identify those patients whose final pathology report may ultimately harbor NIFTP and can be offered a conservative surgical plan to assist in cost-effective, optimal management of patients with NIFTP.

Causes of death among cancer patients.

Background: The purpose of our study was to characterize the causes of death among cancer patients as a function of objectives: (i) calendar year, (ii) patient age, and (iii) time after diagnosis. Patients and methods: US death certificate data in Surveillance, Epidemiology, and End Results Stat 8.2.1 were used to categorize cancer patient death as being due to index-cancer, nonindex-cancer, and noncancer cause from 1973 to 2012. In addition, data were characterized with standardized mortality ratios (SMRs), which provide the relative risk of death compared with all persons. Results: The greatest relative decrease in index-cancer death (generally from > 60% to < 30%) was among those with cancers of the testis, kidney, bladder, endometrium, breast, cervix, prostate, ovary, anus, colorectum, melanoma, and lymphoma. Index-cancer deaths were stable (typically >40%) among patients with cancers of the liver, pancreas, esophagus, and lung, and brain. Noncancer causes of death were highest in patients with cancers of the colorectum, bladder, kidney, endometrium, breast, prostate, testis; >40% of deaths from heart disease. The highest SMRs were from nonbacterial infections, particularly among <50-year olds (e.g. SMR >1,000 for lymphomas, P < 0.001). The highest SMRs were typically within the first year after cancer diagnosis (SMRs 10-10,000, P < 0.001). Prostate cancer patients had increasing SMRs from Alzheimer's disease, as did testicular patients from suicide. Conclusion: The risk of death from index- and nonindex-cancers varies widely among primary sites. Risk of noncancer deaths now surpasses that of cancer deaths, particularly for young patients in the year after diagnosis.

Pathology-based staging for HPV-positive squamous carcinoma of the oropharynx.

OBJECTIVE: The rapid worldwide rise in incidence of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has generated studies confirming this disease as an entity distinct from traditional OPSCC. Based on pathology, surgical studies have revealed prognosticators specific to HPV-positive OPSCC. The current AJCC/UICC staging and pathologic nodal (pN)-classification do not differentiate for survival, demonstrating the need for new, HPV-specific OPSCC staging. The objective of this study was to define a pathologic staging system specific to HPV-positive OPSCC. METHODS: Data were assembled from a surgically-managed, p16-positive OPSCC cohort (any T, any N, M0) of 704 patients from five cancer centers. Analysis was performed for (a) the AJCC/UICC pathologic staging, (b) newly published clinical staging for non-surgically managed HPV-positive OPSCC, and (c) a novel, pathology-based, "HPVpath" staging system that combines features of the primary tumor and nodal metastases. RESULTS: A combination of AJCC/UICC pT-classification and pathology-confirmed metastatic node count (⩽4 versus ⩾5) yielded three groups: stages I (pT1-T2, ⩽4 nodes), II (pT1-T2, ⩾5 nodes; pT3-T4, ⩽4 nodes), and III (pT3-T4, ⩾5 nodes), with incrementally worse prognosis (Kaplan-Meier overall survival of 90%, 84% and 48% respectively). Existing AJCC/UICC pathologic staging lacked prognostic definition. Newly published HPV-specific clinical stagings from non-surgically managed patients, although prognostic, showed lower precision for this surgically managed cohort. CONCLUSIONS: Three loco-regional "HPVpath" stages are identifiable for HPV-positive OPSCC, based on a combination of AJCC/UICC primary tumor pT-classification and metastatic node count. A workable, pathologic staging system is feasible to establish prognosis and guide adjuvant therapy decisions in surgically-managed HPV-positive OPSCC.

Induction cetuximab, paclitaxel, and carboplatin followed by chemoradiation with cetuximab, paclitaxel, and carboplatin for stage III/IV head and neck squamous cancer: a phase II ECOG-ACRIN trial (E2303).

BACKGROUND: E2303 evaluated cetuximab, paclitaxel, and carboplatin used as induction therapy and concomitant with radiation therapy in patients with stage III/IV head and neck squamous cell carcinoma (HNSCC) determining pathologic complete response (CR), event-free survival (EFS), and toxicity. PATIENTS AND METHODS: Patients with resectable stage III/IV HNSCC underwent induction therapy with planned primary site restaging biopsies (at week 8 in clinical complete responders and at week 14 if disease persisted). Chemoradiation (CRT) began week 9. If week 14 biopsy was negative, patients completed CRT (68-72 Gy); otherwise, resection was carried out. p16 protein expression status was correlated with response/survival. RESULTS: Seventy-four patients were enrolled; 63 were eligible. Forty-four (70%) were free of surgery to the primary site, progression, and death 1-year post-treatment. Following induction, 41 (23 CR) underwent week 8 primary site biopsy and 24 (59%) had no tumor (pathologic CR). Week 14 biopsy during chemoradiation (50 Gy) in 34 (15 previously positive biopsy; 19 no prior biopsy) was negative in 33. Thus 90% of eligible patients completed CRT. Overall survival and EFS were 78% and 55% at 3 years, respectively. Disease progression in 23 patients (37%) was local only in 10 (16%), regional in 5 (8%), local and regional in 2 (3%), and distant in 5 patients (8%). There were no treatment-related deaths. Toxicity was primarily hematologic or radiation-related. p16 AQUA score was not associated with response/survival. CONCLUSIONS: Induction cetuximab, paclitaxel, and carboplatin followed by the same drug CRT is safe and induces high primary site response and promising survival. CLINICAL TRIALS NUMBER: NCT 00089297.

Identification of Desulfobacterales as primary hydrogenotrophs in a complex microbial mat community.

Hypersaline microbial mats have been shown to produce significant quantities of H2 under dark, anoxic conditions via cyanobacterial fermentation. This flux of a widely accessible microbial substrate has potential to significantly influence the ecology of the mat, and any consumption will affect the net efflux of H2 that might otherwise be captured as a resource. Here, we focus on H2 consumption in a microbial mat from Elkhorn Slough, California, USA, for which H2 production has been previously characterized. Active biologic H2 consumption in this mat is indicated by a significant time-dependent decrease in added H2 compared with a killed control. Inhibition of sulfate reduction, as indicated by a decrease in hydrogen sulfide production relative to controls, resulted in a significant increase in H2 efflux, suggesting that sulfate-reducing bacteria (SRB) are important hydrogenotrophs. Low methane efflux under these same conditions indicated that methanogens are likely not important hydrogenotrophs. Analyses of genes and transcripts that encode for rRNA or dissimilatory sulfite reductase, using both PCR-dependent and PCR-independent metatranscriptomic sequencing methods, demonstrated that Desulfobacterales are the dominant, active SRB in the upper, H2-producing layer of the mat (0-2 mm). This hypothesis was further supported by the identification of transcripts encoding hydrogenases derived from Desulfobacterales capable of H2 oxidation. Analysis of molecular data provided no evidence for the activity of hydrogenotrophic methanogens. The combined biogeochemical and molecular data strongly indicate that SRB belonging to the Desulfobacterales are the quantitatively important hydrogenotrophs in the Elkhorn Slough mat.

Twelve testable hypotheses on the geobiology of weathering.

Critical Zone (CZ) research investigates the chemical, physical, and biological processes that modulate the Earth's surface. Here, we advance 12 hypotheses that must be tested to improve our understanding of the CZ: (1) Solar-to-chemical conversion of energy by plants regulates flows of carbon, water, and nutrients through plant-microbe soil networks, thereby controlling the location and extent of biological weathering. (2) Biological stoichiometry drives changes in mineral stoichiometry and distribution through weathering. (3) On landscapes experiencing little erosion, biology drives weathering during initial succession, whereas weathering drives biology over the long term. (4) In eroding landscapes, weathering-front advance at depth is coupled to surface denudation via biotic processes. (5) Biology shapes the topography of the Critical Zone. (6) The impact of climate forcing on denudation rates in natural systems can be predicted from models incorporating biogeochemical reaction rates and geomorphological transport laws. (7) Rising global temperatures will increase carbon losses from the Critical Zone. (8) Rising atmospheric P(CO2) will increase rates and extents of mineral weathering in soils. (9) Riverine solute fluxes will respond to changes in climate primarily due to changes in water fluxes and secondarily through changes in biologically mediated weathering. (10) Land use change will impact Critical Zone processes and exports more than climate change. (11) In many severely altered settings, restoration of hydrological processes is possible in decades or less, whereas restoration of biodiversity and biogeochemical processes requires longer timescales. (12) Biogeochemical properties impart thresholds or tipping points beyond which rapid and irreversible losses of ecosystem health, function, and services can occur.

PET CT imaging in patients undergoing sentinel node biopsy for melanoma.

AIMS: Sentinel lymph node biopsy (SLNB) has been adopted in the surgical treatment of melanoma to reduce morbidity and enhance staging. Positron emission tomography with computerised tomography (PET/CT) has been utilised in the staging of patients with malignancy though the role of this imaging modality in early stage melanoma is unclear. This study examined the preoperative value of PET/CT in patients undergoing SLNB for malignant melanoma. METHODS: Patients presenting with primary melanoma without evidence of either locoregional or systemic metastasis were considered candidates for SLNB. Selected patients underwent preoperative PET/CT followed by definitive surgical therapy including SLNB with regional lymphadenectomy, where indicated. RESULTS: During a 12-month period 83 patients were identified as having undergone SLNB for melanoma, of which 37 (45%) had preoperative PET/CT. Mean melanoma thickness 1.9 mm and 2.4 mm (PET/CT vs. no PET/CT, p>0.05). 13 (15.6%) patients were found to have lymphatic metastasis at SLNB; nine of these patients underwent PET/CT, only two of these scans were suggestive of lymphatic metastasis (positive predictive value 24%, negative predictive value 76%). PET/CT revealed no unheralded metastatic disease but did identify a second occult malignancy in 4 (10.8%) patients undergoing therapy for melanoma. CONCLUSIONS: The results of this study do not support the use of PET/CT in patients undergoing SLNB for melanoma. SLNB appears to be a more sensitive staging modality in the detection of lymphatic metastasis; however PET/CT may have a future role as a screening tool for malignancy.

Redox fluctuation structures microbial communities in a wet tropical soil.

Frequent high-amplitude redox fluctuation may be a strong selective force on the phylogenetic and physiological composition of soil bacterial communities and may promote metabolic plasticity or redox tolerance mechanisms. To determine effects of fluctuating oxygen regimens, we incubated tropical soils under four treatments: aerobic, anaerobic, 12-h oxic/anoxic fluctuation, and 4-day oxic/anoxic fluctuation. Changes in soil bacterial community structure and diversity were monitored with terminal restriction fragment length polymorphism (T-RFLP) fingerprints. These profiles were correlated with gross N cycling rates, and a Web-based phylogenetic assignment tool was used to infer putative community composition from multiple fragment patterns. T-RFLP ordinations indicated that bacterial communities from 4-day oxic/anoxic incubations were most similar to field communities, whereas those incubated under consistently aerobic or anaerobic regimens developed distinctly different molecular profiles. Terminal fragments found in field soils persisted either in 4-day fluctuation/aerobic conditions or in anaerobic/12-h treatments but rarely in both. Only 3 of 179 total fragments were ubiquitous in all soils. Soil bacterial communities inferred from in silico phylogenetic assignment appeared to be dominated by Actinobacteria (especially Micrococcus and Streptomycetes), "Bacilli," "Clostridia," and Burkholderia and lost significant diversity under consistently or frequently anoxic incubations. Community patterns correlated well with redox-sensitive processes such as nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and denitrification but did not predict patterns of more general functions such as N mineralization and consumption. The results suggest that this soil's indigenous bacteria are highly adapted to fluctuating redox regimens and generally possess physiological tolerance mechanisms which allow them to withstand unfavorable redox periods.

Preliminary results of Radiation Therapy Oncology Group 97-03: a randomized phase ii trial of concurrent radiation and chemotherapy for advanced squamous cell carcinomas of the head and neck.

PURPOSE: To define further the role of concurrent chemoradiotherapy for patients with advanced squamous carcinoma of the head and neck. PATIENTS AND METHODS: The Radiation Therapy Oncology Group developed this three-arm randomized phase II trial. Patients with stage III or IV squamous carcinoma of the oral cavity, oropharynx, or hypopharynx were eligible. Each of three arms proposed a radiation schedule of 70 Gy in 35 fractions. Patients on arm 1 were to receive cisplatin 10 mg/m(2) daily and fluorouracil (FU) 400 mg/m(2) continuous infusion (CI) daily for the final 10 days of treatment. Treatment on arm 2 consisted of hydroxyurea 1 g every 12 hours and FU 800 mg/m(2)/d CI delivered with each fraction of radiation. Arm 3 patients were to receive weekly paclitaxel 30 mg/m(2) and cisplatin 20 mg/m(2). Patients randomly assigned to arms 1 and 3 were to receive their treatments every week; patients on arm 2 were to receive their therapy every other week. RESULTS: Between 1997 and 1999, 241 patients were entered onto study; 231 were analyzable. Ninety-two percent, 79%, and 83% of patients on arms 1, 2, and 3, respectively, were able to complete their radiation as planned or with an acceptable variation. Fewer than 10% of patients had unacceptable deviations or incomplete chemotherapy in the three arms. Estimated 2-year disease-free and overall survival rates were 38.2% and 57.4% for arm 1, 48.6% and 69.4% for arm 2, and 51.3% and 66.6% for arm 3. CONCLUSION: We have demonstrated that three different approaches of concurrent multiagent chemotherapy and radiation were feasible and could be delivered to patients in a multi-institutional setting with high compliance rates.

Gastrostomy-site tumor recurrence is not always fatal.

Percutaneous gastrostomy (PEG) site tumor seeding is a rare occurrence with fewer than 30 reported cases. Reported treatment ranges from observation to wide excision with unanimously poor outcome. We report the first published case of a long-term survivor after resection of a PEG site recurrence and review our treatment approach.

Elevated furin expression in aggressive human head and neck tumors and tumor cell lines.

Pro-protein convertases (PCs) are proteases that recognize and cleave precursor proteins. Furin, a well-studied PC, is ubiquitously expressed, and it has been implicated in many physiological and pathological processes. Some substrates for furin, such as membrane type 1 (MT1) matrix metalloproteinase (MMP), an MMP that activates gelatinase, a collagen-degrading enzyme, are associated with the advanced malignant phenotype. This report examines the expression of furin in carcinoma cell lines of different invasive ability. The levels of furin mRNA and protein correlated with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. Furin expression also was investigated in primary head and neck squamous cell carcinomas (HNSCCs). Furin mRNA was not detected in nonmetastasizing carcinomas. In contrast, furin mRNA was expressed in metastasizing HNSCCs. Immunohistochemistry and Western blot analysis confirmed these results at the protein level. Furin activity was investigated indirectly by evaluating the expression of the pro-form and the processed form of MT1-MMP. Metastasizing HNSCCs showed increased expression of MT1-MMP. Furthermore, pro-MT1-MMP expression was noted in most of the nonmetastasizing HNSCCs analyzed by Western blot, and it was absent in the metastasizing HNSCCs. This finding suggests a lower level of furin-mediated MT1-MMP activation in the less aggressive cancers. These observations indicate that furin plays a role in tumor progression. Its overexpression in more aggressive or metastasizing cancers resulted in increased MMP processing.

Image cytometry of cyclin D1: a prognostic marker for head and neck squamous cell carcinomas.

CCND1 gene amplification and cyclin D1 protein overexpression are indicators for poor prognosis in invasive head and neck carcinomas. Increased CCND1 gene dosage is a more sensitive prognostic factor than protein overexpression as evaluated by conventional immunohistochemical techniques. Qualitative immunohistochemistry cannot distinguish cyclin D1 overexpression accompanied by amplification of the CCND1 gene from overexpression associated with normal CCND1 gene copy number. To improve the sensitivity of cyclin D1 protein determination, we applied quantitative techniques of image analysis to evaluate cyclin D1 in 54 head and neck carcinomas. There was a significantly higher rate of occurrence of adverse events (P = 0.043) among patients with CCND1 gene amplification than among those without gene amplification. There was a strong association between CCND1 gene amplification (as detected by Southern blot analysis) and the highest nuclear score (by image cytometry of the immunostained tumor sections). The predominance of cells in the lowest nuclear score category was significantly associated with normal copy number (P = 0.005). Conversely, the highest nuclear score was a significant predictor of gene dosage (P = 0.02). Similarly, high nuclear score was a good predictor of death as the final outcome of the disease (P = 0.01). Although somewhat less accurate than Southern blotting, image cytometry of immunohistochemical cyclin D1 stain appears to be a promising tool that could be useful for other tumor marker expression studies.

Incidental parathyroidectomy during thyroid surgery does not cause transient symptomatic hypocalcemia.

OBJECTIVES: To identify any risk factors for incidental parathyroidectomy and to define its association with symptomatic postoperative hypocalcemia. DESIGN: Retrospective study. SETTING: Tertiary referral cancer center. PATIENTS: Consecutive patients who underwent thyroid surgery between 1991 and 1999. Patients who underwent procedures for locally advanced thyroid cancer requiring laryngectomy, tracheal resection, or esophagectomy were excluded. INTERVENTIONS: All pathology reports were reviewed for the presence of any parathyroid tissue in the resected specimen. Slides were reviewed, and information regarding patient demographics, diagnosis, operative details, and postoperative complications was collected. MAIN OUTCOME MEASURE: Identification of parathyroid tissue in resected specimens and postoperative symptomatic hypocalcemia. RESULTS: A total of 141 thyroid procedures were performed: 69 total thyroidectomies (49%) and 72 total thyroid lobectomies (51%). The findings were benign in 68 cases (48%) and malignant in 73 cases (52%). In the entire series, incidental parathyroidectomy was found in 21 cases (15%). Parathyroid tissue was found in intrathyroidal (50%), extracapsular (31%), and central node compartment (19%) sites. The performance of a concomitant modified radical neck dissection was associated with an increased risk of unplanned parathyroidectomy (P =.05). There was no association of incidental parathyroidectomy with postoperative hypocalcemia (P =.99). Multivariate analysis identified total thyroidectomy as a risk factor for postoperative hypocalcemia (P =.008). In the entire study group, transient symptomatic hypocalcemia occurred in 9 patients (6%), and permanent hypocalcemia occurred in 1 patient who underwent a total thyroidectomy and concomitant neck dissection. CONCLUSIONS: Unintended parathyroidectomy, although not uncommon, is not associated with symptomatic postoperative hypocalcemia. Modified radical neck dissection may increase the risk of incidental parathyroidectomy. Most of the glands removed were intrathyroidal, so changes in surgical technique are unlikely to markedly reduce this risk.

Testing time-, ignorance-, and danger-based models of tolerance.

In this study, we present data showing that tolerance to Ags in the periphery is not determined by the time at which the Ag appears, or by special properties of tissues in newborn mice or newly developing immune systems. We placed male grafts onto immunoincompetent female mice, allowed the grafts to heal for up to 5 mo, and then repopulated the recipients with fetal liver stem cells. We found that the newly arising T cells were neither tolerant nor ignorant of the grafts, but promptly rejected them, though they did not reject female grafts, nor show any signs of autoimmunity. We also found that the H-Y Ag was continuously cross-presented on host APCs, that this presentation was immunogenic, not tolerogenic, and that it depended on the continuous presence of the graft. In searching for the stimulus that might activate the host APCs, we analyzed mRNA expression with a highly sensitive real-time quantitative PCR assay. By using two different "housekeeping" molecules for comparison, we analyzed the message levels for several stress and/or inflammatory molecules in the healed grafts. We found that the long-healed grafts were not equivalent to "normal" skin because the healed grafts expressed lower levels of GAPDH. Altogether, these data suggest that acceptance vs rejection of peripheral tissues is not attributable to ignorance, timing-based tolerance, or special circulation properties of naive T cells in neonatal tissues. It is more likely attributable to an aspect of the context of Ag presentation that remains to be identified.

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy.

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.

Ubiquinone binding, ubiquinone exclusion, and detailed cofactor conformation in a mutant bacterial reaction center.

The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.