Background Antenatal care (ANC) presents a potentially valuable platform for integrated delivery of additional health services for pregnant women-services that are vital to reduce the persistently high rates of maternal and neonatal mortality in low-and middle-income countries (LMICs). However, there is limited evidence on the impact of integrating health services with ANC to guide policy. This review assesses the impact of integration of postnatal and other health services with ANC on health services uptake and utilisation, health outcomes and user experience of care in LMICs.Methods Cochrane Library, MEDLINE, Embase, CINAHL Plus, POPLINE and Global Health were searched for studies that compared integrated models for delivery of postnatal and other health services with ANC to non-integrated models. Risk of bias of included studies was assessed using the Cochrane Effective Practice and Organisation of Care (EPOC) criteria and the Newcastle-Ottawa Scale, depending on the study design. Due to high heterogeneity no meta-analysis could be conducted. Results are presented narratively.Findings 12 studies were included in the review. Limited evidence, with moderate-to high-risk of bias, suggests that integrated service delivery results in improved uptake of essential health services for women, earlier initiation of treatment, and better health outcomes. Women also reported improved satisfaction with integrated services.Conclusions The reported evidence is largely based on non-randomised studies with poor generalizability, and therefore offers very limited policy guidance. More rigorously conducted and geographically diverse studies are needed to better ascertain and quantify the health and economic benefits of integrating health services with ANC.

Background Since 2002, development assistance for health has substantially increased, especially investments for HIV, tuberculosis (TB) and malaria control. We undertook a systematic review to assess and synthesize the existing evidence in the scientific literature on the health impacts of these investments. Methods and findings We systematically searched databases for peer-reviewed and grey literature, using tailored search strategies. We screened studies for study design and relevance, using predefined inclusion criteria, and selected those that enabled us to link health outcomes or impact to increased external funding. For all included studies, we recorded dataset and study characteristics, health outcomes and impacts. We analysed the data using a causal-chain framework to develop a narrative summary of the published evidence. Thirteen articles, representing 11 individual studies set in Africa and Asia reporting impacts on HIV, tuberculosis and malaria, met the inclusion criteria. Only two of these studies documented the entire causal-chain spanning from funding to programme scale-up, to outputs, outcomes and impacts. Nonetheless, overall we find a positive correlation between consecutive steps in the causal chain, suggesting that external funds for HIV, tuberculosis and malaria programmes contributed to improved health outcomes and impact. Conclusions Despite the large number of supported programmes worldwide and despite an abundance of published studies on HIV, TB and malaria control, we identified very few eligible studies that adequately demonstrated the full process by which external funding has been translated to health impact. Most of these studies did not move beyond demonstrating statistical association, as opposed to contribution or causation. We thus recommend that funding organizations and researchers increase the emphasis on ensuring data capture along the causal pathway to demonstrate effect and contribution of external financing. The findings of these comprehensive and rigorously conducted impact evaluations should also be made publicly accessible.

BackgroundThis review is an update of the original Cochrane review published in July 2012. Missed appointments are a major cause of inefficiency in healthcare delivery with substantial monetary costs for the health system, leading to delays in diagnosis and appropriate treatment. Patients' forgetfulness is one of the main reasons for missed appointments. Patient reminders may help reduce missed appointments. Modes of communicating reminders for appointments to patients include face-to-face communication, postal messages, calls to land-lines or mobile phones, and mobile phone messaging. Mobile phone messaging applications, such as Short Message Service (SMS) and Multimedia Message Service (MMS), could provide an important, inexpensive delivery medium for reminders for healthcare appointments.ObjectivesTo update our review assessing the effects of mobile phone messaging reminders for attendance at healthcare appointments. Secondary objectives include assessment of costs; health outcomes; patients' and healthcare providers' evaluation of the intervention and perceptions of safety; and possible harms and adverse effects associated with the intervention.Search methodsOriginal searches were run in June 2009. For this update, we searched the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library 2012, Issue 8), MEDLINE (OvidSP) (January 1993 to August 2012), EMBASE (OvidSP) (January 1993 to August 2012), PsycINFO (OvidSP) (January 1993 to August 2012) and CINAHL (EbscoHOST) (January 1993 to August 2012). We also reviewed grey literature (including trial registers) and reference lists of articles.Selection criteriaRandomised controlled trials (RCTs) assessing mobile phone messaging as reminders for healthcare appointments. We only included studies in which it was possible to assess effects of mobile phone messaging independent of other technologies or interventions.Data collection and analysisTwo review authors independently assessed all studies against the inclusion criteria, with any disagreements resolved by a third review author. Study design features, characteristics of target populations, interventions and controls, and results data were extracted by two review authors and confirmed by a third author. Two authors assessed the risk of bias of the included studies. As the intervention characteristics and outcome measures were similar across included studies, we conducted a meta-analysis to estimate an overall effect size.Main resultsWe included eight randomised controlled trials involving 6615 participants. Four of these studies were newly identified during this update.We found moderate quality evidence from seven studies (5841 participants) that mobile text message reminders improved the rate of attendance at healthcare appointments compared to no reminders (risk ratio (RR) 1.14 (95% confidence interval (CI) 1.03 to 1.26)). There was also moderate quality evidence from three studies (2509 participants) that mobile text message reminders had a similar impact to phone call reminders (RR 0.99 (95% CI 0.95 to 1.02). Low quality evidence from one study (291 participants) suggests that mobile text message reminders combined with postal reminders improved the rate of attendance at healthcare appointments compared to postal reminders alone (RR 1.10 (95% CI 1.02 to 1.19)). Overall, the attendance to appointment rates were 67.8% for the no reminders group, 78.6% for the mobile phone messaging reminders group and 80.3% for the phone call reminders group. One study reported generally that there were no adverse effects during the study period; none of the studies reported in detail on specific adverse events such as loss of privacy, data misinterpretation, or message delivery failure. Two studies reported that the costs per text message per attendance were respectively 55% and 65% lower than costs per phone call reminder. The studies included in the review did not report on health outcomes or people's perceptions of safety related to receiving reminders by text message.Authors' conclusionsLow to moderate quality evidence included in this review shows that mobile phone text messaging reminders increase attendance at healthcare appointments compared to no reminders, or postal reminders.Text messaging reminders were similar to telephone reminders in terms of their effect on attendance rates, and cost less than telephone reminders. However, the included studies were heterogeneous and the quality of the evidence therein is low to moderate. Further, there is a lack of information about health effects, adverse effects and harms, user evaluation of the intervention and user perceptions of its safety. The current evidence therefore still remains insufficient to conclusively inform policy decisions.There is a need for more high-quality randomised trials of mobile phone messaging reminders, that measure not only patients' attendance rates, but also focus on the cost-effectiveness of these interventions. Health outcomes, patients' and healthcare providers' evaluation and perceptions of the safety of the interventions, potential harms, and adverse effects of mobile phone messaging reminders should be assessed. Studies should report message content and timing in relation to the appointment.

BackgroundPreventive health care promotes health and prevents disease or injuries by addressing factors that lead to the onset of a disease, and by detecting latent conditions to reduce or halt their progression. Many risk factors for costly and disabling conditions (such as cardiovascular diseases, cancer, diabetes, and chronic respiratory diseases) can be prevented, yet healthcare systems do not make the best use of their available resources to support this process. Mobile phone messaging applications, such as Short Message Service (SMS) and Multimedia Message Service (MMS), could offer a convenient and cost-effective way to support desirable health behaviours for preventive health care.ObjectivesTo assess the effects of mobile phone messaging interventions as a mode of delivery for preventive health care, on health status and health behaviour outcomes.Search methodsWe searched: the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library 2009, Issue 2), MEDLINE (OvidSP) (January 1993 to June 2009), EMBASE (OvidSP) (January 1993 to June 2009), PsycINFO (OvidSP) (January 1993 to June 2009), CINAHL (EbscoHOST) (January 1993 to June 2009), LILACS (January 1993 to June 2009) and African Health Anthology (January 1993 to June 2009).We also reviewed grey literature (including trial registers) and reference lists of articles.Selection criteriaWe included randomised controlled trials (RCTs), quasi-randomised controlled trials (QRCTs), controlled before-after (CBA) studies, and interrupted time series (ITS) studies with at least three time points before and after the intervention. We included studies using SMS or MMS as a mode of delivery for any type of preventive health care. We only included studies in which it was possible to assess the effects of mobile phone messaging independent of other technologies or interventions.Data collection and analysisTwo review authors independently assessed all studies against the inclusion criteria, with any disagreements resolved by a third review author. Study design features, characteristics of target populations, interventions and controls, and results data were extracted by two review authors and confirmed by a third author. Primary outcomes of interest were health status and health behaviour outcomes. We also considered patients' and providers' evaluation of the intervention, perceptions of safety, health service utilisation and costs, and potential harms or adverse effects. Because the included studies were heterogeneous in type of condition addressed, intervention characteristics and outcome measures, we did not consider that it was justified to conduct a meta-analysis to derive an overall effect size for the main outcome categories; instead, we present findings narratively.Main resultsWe included four randomised controlled trials involving 1933 participants.For the primary outcome category of health, there was moderate quality evidence from one study that women who received prenatal support via mobile phone messages had significantly higher satisfaction than those who did not receive the messages, both in the antenatal period (mean difference (MD) 1.25, 95% confidence interval (CI) 0.78 to 1.72) and perinatal period (MD 1.19, 95% CI 0.37 to 2.01). Their confidence level was also higher (MD 1.12, 95% CI 0.51 to 1.73) and anxiety level was lower (MD -2.15, 95% CI -3.42 to -0.88) than in the control group in the antenatal period. In this study, no further differences were observed between groups in the perinatal period. There was low quality evidence that the mobile phone messaging intervention did not affect pregnancy outcomes (gestational age at birth, infant birth weight, preterm delivery and route of delivery).For the primary outcome category of health behaviour, there was moderate quality evidence from one study that mobile phone message reminders to take vitamin C for preventive reasons resulted in higher adherence (risk ratio (RR) 1.41, 95% CI 1.14 to 1.74). There was high quality evidence from another study that participants receiving mobile phone messaging support had a significantly higher likelihood of quitting smoking than those in a control group at 6 weeks (RR 2.20, 95% CI 1.79 to 2.70) and at 12 weeks follow-up (RR 1.55, 95% CI 1.30 to 1.84). At 26 weeks, there was only a significant difference between groups if, for participants with missing data, the last known value was carried forward. There was very low quality evidence from one study that mobile phone messaging interventions for self-monitoring of healthy behaviours related to childhood weight control did not have a statistically significant effect on physical activity, consumption of sugar-sweetened beverages or screen time.For the secondary outcome of acceptability, there was very low quality evidence from one study that user evaluation of the intervention was similar between groups. There was moderate quality evidence from one study of no difference in adverse effects of the intervention, measured as rates of pain in the thumb or finger joints, and car crash rates.None of the studies reported the secondary outcomes of health service utilisation or costs of the intervention.Authors' conclusionsWe found very limited evidence that in certain cases mobile phone messaging interventions may support preventive health care, to improve health status and health behaviour outcomes. However, because of the low number of participants in three of the included studies, combined with study limitations of risk of bias and lack of demonstrated causality, the evidence for these effects is of low to moderate quality. The evidence is of high quality only for interventions aimed at smoking cessation. Furthermore, there are significant information gaps regarding the long-term effects, risks and limitations of, and user satisfaction with, such interventions.

BackgroundLong-term illnesses affect a significant proportion of the population in developed and developing countries. Mobile phone messaging applications, such as Short Message Service (SMS) and Multimedia Message Service (MMS), may present convenient, cost-effective ways of supporting self-management and improving patients' self-efficacy skills through, for instance, medication reminders, therapy adjustments or supportive messages.ObjectivesTo assess the effects of mobile phone messaging applications designed to facilitate self-management of long-term illnesses, in terms of impact on health outcomes and patients' capacity to self-manage their condition. Secondary objectives include assessment of: user evaluation of the intervention; health service utilisation and costs; and possible risks and harms associated with the intervention.Search methodsWe searched the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library 2009, Issue 2), MEDLINE (OvidSP) (January 1993 to June 2009), EMBASE (OvidSP) (January 1993 to June 2009), PsycINFO (OvidSP) (January 1993 to June 2009), CINAHL (EbscoHOST) (January 1993 to June 2009), LILACS (January 1993 to June 2009) and African Health Anthology (January 1993 to June 2009). We also reviewed grey literature (including trial registers) and reference lists of articles.Selection criteriaWe included randomised controlled trials (RCTs), quasi-randomised controlled trials (QRCTs), controlled before-after (CBA) studies, or interrupted time series (ITS) studies with at least three time points before and after the intervention. We selected only studies where it was possible to assess the effects of mobile phone messaging independent of other technologies or interventions.Data collection and analysiswo review authors independently assessed all studies against the inclusion criteria, with any disagreements resolved by a third review author. Study design features, characteristics of target populations, interventions and controls, and results data were extracted by two review authors and confirmed by a third. Primary outcomes of interest were health outcomes as a result of the intervention and capacity to self-manage long-term conditions. We also considered patients' and providers' evaluation of the intervention, perceptions of safety, health service utilisation and costs, and potential harms or adverse effects. The included studies were heterogeneous in type of condition addressed, intervention characteristics and outcome measures. Therefore, a meta-analysis to derive an overall effect size for the main outcome categories was not considered justified and findings are presented narratively.Main resultsWe included four randomised controlled trials involving 182 participants.For the primary outcome of health outcomes, including physiological measures, there is moderate quality evidence from two studies involving people with diabetes showing no statistical difference from text messaging interventions compared with usual care or email reminders for glycaemic control (HbA1c), the frequency of diabetic complications, or body weight. There is moderate quality evidence from one study of hypertensive patients that the mean blood pressure and the proportion of patients who achieved blood pressure control were not significantly different in the intervention and control groups, and that there was no statistically significant difference in mean body weight between the groups. There is moderate quality evidence from one study that asthma patients receiving a text messaging intervention experienced greater improvements on peak expiratory flow variability (mean difference (MD) -11.12, 95% confidence interval (CI) -19.56 to -2.68) and the pooled symptom score comprising four items (cough, night symptoms, sleep quality, and maximum tolerated activity) (MD -0.36, 95% CI -0.56 to -0.17) compared with the control group. However, the study found no significant differences between the groups in impact on forced vital capacity or forced expiratory flow in 1 second.For the primary outcome of capacity to self-manage the condition, there is moderate quality evidence from one study that diabetes patients receiving the text messaging intervention demonstrated improved scores on measures of self-management capacity (Self-Efficacy for Diabetes score (MD 6.10, 95% CI 0.45 to 11.75), Diabetes Social Support Interview pooled score (MD 4.39, 95% CI 2.85 to 5.92)), but did not show improved knowledge of diabetes. There is moderate quality evidence from three studies of the effects on treatment compliance. One study showed an increase in hypertensive patients' rates of medication compliance in the intervention group (MD 8.90, 95% CI 0.18 to 17.62) compared with the control group, but in another study there was no statistically significant effect on rates of compliance with peak expiratory flow measurement in asthma patients. Text message prompts for diabetic patients initially also resulted in a higher number of blood glucose results sent back (46.0) than email prompts did (23.5).For the secondary outcome of participants' evaluation of the intervention, there is very low quality evidence from two studies that patients receiving mobile phone messaging support reported perceived improvement in diabetes self-management, wanted to continue receiving messages, and preferred mobile phone messaging to email as a method to access a computerised reminder system.For the secondary outcome of health service utilisation, there is very low quality evidence from two studies. Diabetes patients receiving text messaging support made a comparable number of clinic visits and calls to an emergency hotline as patients without the support. For asthma patients the total number of office visits was higher in the text messaging group, whereas the number of hospital admissions was higher for the control group.Because of the small number of trials included, and the low overall number of participants, for any of the reviewed outcomes the quality of the evidence can at best be considered moderate.Authors' conclusionsWe found some, albeit very limited, indications that in certain cases mobile phone messaging interventions may provide benefit in supporting the self-management of long-term illnesses. However, there are significant information gaps regarding the long-term effects, acceptability, costs, and risks of such interventions. Given the enthusiasm with which so-called mHealth interventions are currently being implemented, further research into these issues is needed.

BackgroundMobile phone messaging, such as Short Message Service (SMS) and Multimedia Message Service (MMS), has rapidly grown into a mode of communication with a wide range of applications, including communicating the results from medical investigations to patients. Alternative modes of communication of results include face-to-face communication, postal messages, calls to landlines or mobile phones, through web-based health records and email. Possible advantages of mobile phone messaging include convenience to both patients and healthcare providers, reduced waiting times for health services and healthcare costs.ObjectivesTo assess the effects of mobile phone messaging for communicating results of medical investigations, on people's healthcare-seeking behaviour and health outcomes. Secondary objectives include assessment of participants' evaluation of the intervention, direct and indirect healthcare costs and possible risks and harms associated with the intervention.Search methodsWe searched the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library 2009, Issue 2), MEDLINE (OvidSP) (January 1993 to June 2009), EMBASE (OvidSP) (January 1993 to June 2009), PsycINFO (OvidSP) (January 1993 to June 2009), CINAHL (EbscoHOST) (January 1993 to June 2009), LILACS (January 1993 to June 2009) and African Health Anthology (January 1993 to June 2009). We also reviewed grey literature (including trial registers) and reference lists of articles.Selection criteriaWe included randomised controlled trials (RCTs), quasi-randomised controlled trials (QRCTs), controlled before-after (CBA) studies, or interrupted time series (ITS) studies with at least three time points before and after the intervention. We included studies assessing mobile phone messaging for communicating results of medical tests, between a healthcare provider or 'treatment buddy' and patient. We only included studies in which it was possible to assess the effects of mobile phone messaging independent of other technologies or interventions.Data collection and analysisTwo review authors independently assessed all studies against the inclusion criteria, with any disagreements resolved by a third review author. Study design features, characteristics of target populations, interventions and controls, and results data were extracted by two review authors and confirmed by a third. Primary outcomes of interest were health outcomes and healthcare utilisation as a result of the intervention. We also considered patients' and providers' evaluation of the intervention, perceptions of safety, costs and potential harms or adverse effects of the intervention.Main resultsWe included one randomised controlled trial involving 2782 participants. The study investigated the effects of mobile phone messaging in alleviating anxiety in women waiting for prenatal biochemical screening results for Down syndrome, by providing fast reporting of results before a follow-up appointment. The study measured health outcomes using the Spielberger State-Trait Anxiety Inventory (STAI), which includes a scale (20 to 80 points, higher score indicates higher anxiety) to describe how the respondent feels at a particular moment in time (state anxiety). The study, which was at high risk of bias, found that women who had received their test result early by text message had a mean anxiety score 2.48 points lower than women who had not yet received their result (95% CI -8.79 to 3.84). Women with a serum-negative test result receiving their result early had a mean anxiety score 5.3 points lower (95% CI -5.99 to -4.61) than women in the control group. Women with a serum-positive test result receiving their result early by text message had a mean anxiety score 1.2 points higher (95% CI -3.48 to 5.88) than women in the control group. The evidence was of low quality due to high risk of bias in the included study, and the fact that the evidence comes from one study only. The study did not report on other outcomes of interest, such as patient satisfaction, adverse events or cost.Authors' conclusionsWe found very limited evidence of low quality that communicating results of medical investigations by mobile phone messaging may make little or no difference to women's anxiety overall or in women with positive test results, but may reduce anxiety in women with negative test results. However, with only one study included in this review, this evidence is insufficient to inform recommendations at this time. More research is needed on the effectiveness and user evaluation of these interventions. In particular, more research should be conducted into the potential risks and limitations of these interventions.

Background: Objective of the study was to assess the effects of strategies to integrate targeted priority population, health and nutrition interventions into health systems on patient health outcomes and health system effectiveness and thus to compare integrated and non-integrated health programmes.Methods: Systematic review using Cochrane methodology of analysing randomised trials, controlled before-and-after and interrupted time series studies. We defined specific strategies to search PubMed, CENTRAL and the Cochrane Effective Practice and Organisation of Care Group register, considered studies published from January 1998 until September 2008, and tracked references and citations. Two reviewers independently agreed on eligibility, with an additional arbiter as needed, and extracted information on outcomes: primary (improved health, financial protection, and user satisfaction) and secondary (improved population coverage, access to health services, efficiency, and quality) using standardised, pre-piloted forms. Two reviewers in the final stage of selection jointly assessed quality of all selected studies using the GRADE criteria.Results: Of 8,274 citations identified 12 studies met inclusion criteria. Four studies compared the benefits of Integrated Management of Childhood Illnesses in Tanzania and Bangladesh, showing improved care management and higher utilisation of health facilities at no additional cost. Eight studies focused on integrated delivery of mental health and substance abuse services in the United Kingdom and United States of America. Integrated service delivery resulted in better clinical outcomes and greater reduction of substance abuse in specific subgroups of patients, with no significant difference found overall. Quality of care, patient satisfaction, and treatment engagement were higher in integrated delivery models.Conclusions: Targeted priority population health interventions we identified led to improved health outcomes, quality of care, patient satisfaction and access to care. Limited evidence with inconsistent findings across varied interventions in different settings means no general conclusions can be drawn on the benefits or disadvantages of integrated service delivery.

A longstanding debate on health systems organization relates to benefits of integrating health programmes that emphasize specific interventions into mainstream health systems to increase access and improve health outcomes. This debate has long been characterized by polarization of views and ideologies, with protagonists for and against integration arguing the relative merits of each approach. However, all too frequently these arguments have not been based on hard evidence. The presence of both integrated and non-integrated programmes in many countries suggests there may be benefits to either approach, but the relative merits of integration in various contexts and for different interventions have not been systematically analysed and documented. In this paper we present findings of a systematic review that explores a broad range of evidence on: (i) the extent and nature of the integration of targeted health programmes that emphasize specific interventions into critical health systems functions, (ii) how the integration or non-integration of health programmes into critical health systems functions in different contexts has influenced programme success, (iii) how contextual factors have affected the extent to which these programmes were integrated into critical health systems functions. Our analysis shows few instances where there is full integration of a health intervention or where an intervention is completely non-integrated. Instead, there exists a highly heterogeneous picture both for the nature and also for the extent of integration. Health systems combine both non-integrated and integrated interventions, but the balance of these interventions varies considerably.

The benefits of integrating programmes that emphasize specific interventions into health systems to improve health outcomes have been widely debated. This debate has been driven by narrow binary considerations of integrated (horizontal) versus non-integrated (vertical) programmes, and characterized by polarization of views with protagonists for and against integration arguing the relative merits of each approach. The presence of both integrated and non-integrated programmes in many countries suggests benefits to each approach. While the terms 'vertical' and 'integrated' are widely used, they each describe a range of phenomena. In practice the dichotomy between vertical and horizontal is not rigid and the extent of verticality or integration varies between programmes. However, systematic analysis of the relative merits of integration in various contexts and for different interventions is complicated as there is no commonly accepted definition of 'integration'-a term loosely used to describe a variety of organizational arrangements for a range of programmes in different settings. We present an analytical framework which enables deconstruction of the term integration into multiple facets, each corresponding to a critical health system function. Our conceptual framework builds on theoretical propositions and empirical research in innovation studies, and in particular adoption and diffusion of innovations within health systems, and builds on our own earlier empirical research. It brings together the critical elements that affect adoption, diffusion and assimilation of a health intervention, and in doing so enables systematic and holistic exploration of the extent to which different interventions are integrated in varied settings and the reasons for the variation. The conceptual framework and the analytical approach we propose are intended to facilitate analysis in evaluative and formative studies of-and policies on-integration, for use in systematically comparing and contrasting health interventions in a country or in different settings to generate meaningful evidence to inform policy.

A dimer of N42C azurin (P. aeruginosa), formed by intermolecular disulfide bond formation between the introduced surface-exposed cysteine residues, was previously shown to exhibit low rates of intramolecular electron self-exchange. The work presented here aims to render the construct sensitive to pH by the introduction of an additional surface mutation (M64E). The structural and mechanistic effects of ionisable residues in the hydrophobic patch were analysed as a function of pH. A crystal structure of the dimer at pH = 3.1, solved to 2.3 angstrom resolution, displays an orientation distinct from other known azurin complexes. The dimer exhibits slow intramolecular self-exchange at pH* = 4.5 (k(ese)(intra) approximate to 25 s(-1)), in line with the Cu-Cu distance of 20.5 angstrom observed in the crystal structure of the dimer. The intermolecular electron self-exchange rate constant amounts to k(inter) = (1-8 +/- 0-1) x 10(4) m(-1)s(-1). Thus, at protein concentrations less than 1 mM, the intramolecular process dominates the electron self-exchange. A change from low to high pH results in a slowing down of the electron- exchange processes. A partitioning between intra- and intermolecular exchange proved impossible at low pH due to the limited precision of the experimental data. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007).

A dimer of N42C azurin ( P. aeruginosa ), formed by intermolecular disulfide bond formation between the introduced surface‐exposed cysteine residues, was previously shown to exhibit low rates of intramolecular electron self‐exchange. The work presented here aims to render the construct sensitive to pH by the introduction of an additional surface mutation (M64E). The structural and mechanistic effects of ionisable residues in the hydrophobic patch were analysed as a function of pH. A crystal structure of the dimer at pH = 3.1, solved to 2.3 Å resolution, displays an orientation distinct from other known azurin complexes. The dimer exhibits slow intramolecular self‐exchange at pH* = 4.5 ( k ese intra ≈ 25 s –1 ), in line with the Cu–Cu distance of 20.5 Å observed in the crystal structure of the dimer. The intermolecular electron self‐exchange rate constant amounts to k inter = (1.8 ± 0.1) ×10 4 M –1 s –1 . Thus, at protein concentrations less than 1 m m, the intramolecular process dominates the electron self‐exchange. A change from low to high pH results in a slowing down of the electron‐exchange processes. A partitioning between intra‐ and intermolecular exchange proved impossible at low pH due to the limited precision of the experimental data. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Electron paramagnetic resonance (EPR) and Fourier transform IR studies on the soluble hydrogenase from Acidithiobacillus ferrooxidans are presented. In addition, detailed sequence analyses of the two subunits of the enzyme have been performed. They show that the enzyme belongs to a group of uptake [NiFe] hydrogenases typical for Cyanobacteria. The sequences have also a close relationship to those of the H(2)-sensor proteins, but clearly differ from those of standard [NiFe] hydrogenases. It is concluded that the structure of the catalytic centre is similar, but not identical, to that of known [NiFe] hydrogenases. The active site in the majority of oxidized enzyme molecules, 97% in cells and more than 50% in the purified enzyme, is EPR-silent. Upon contact with H(2) these sites remain EPR-silent and show only a limited IR response. Oxidized enzyme molecules with an EPR-detectable active site show a Ni(r)*-like EPR signal which is light-sensitive at cryogenic temperatures. This is a novelty in the field of [NiFe] hydrogenases. Reaction with H(2) converts these active sites to the well-known Ni(a)-C* state. Illumination below 160 K transforms this state into the Ni(a)-L* state. The reversal, in the dark at 200 K, proceeds via an intermediate Ni EPR signal only observed with the H(2)-sensor protein from Ralstonia eutropha. The EPR-silent active sites in as-isolated and H(2)-treated enzyme are also light-sensitive as observed by IR spectra at cryogenic temperatures. The possible origin of the light sensitivity is discussed. This study represents the first spectral characterization of an enzyme of the group of cyanobacterial uptake hydrogenases.