Oxygen kinetics and debt during recovery from expiratory flow-limited exercise in healthy humans

In healthy subjects expiratory flow limitation (EFL) during exercise can lower O2 delivery to the working muscles. We hypothesized that if this affects exercise performance it should influence O2 kinetics at the end of exercise when the O2 debt is repaid. We performed an incremental exercise test on six healthy males with a Starling resistor in the expiratory line limiting expiratory flow to ∼ 1 l s−1 to determine maximal EFL exercise workload (Wmax). In two more square-wave exercise runs subjects exercised with and without EFL at Wmax for 6 min, while measuring arterial O2 saturation (% SaO2), end-tidal pressure of CO2 (PETCO2) and breath-by-breath O2 consumption taking into account changes in O2 stored in the lungs. Over the last minute of EFL exercise, mean PETCO2 (54.7 ± 9.9 mmHg) was significantly higher (P < 0.05) compared to control (41.4 ± 3.9 mmHg). At the end of EFL exercise %SaO2 fell significantly by 4 ± 3%. When exercise stopped, EFL was removed, and we continued to measure During recovery, there was an immediate step increase in so that repayment of EFL O2 debt started at a higher than control. Recovery kinetics after EFL exercise was best characterized by a double-exponential function with fundamental and slow time constants of 27 ± 11 and 1,020 ± 305 s, compared to control values of 41 ± 10 and 1,358 ± 320 s, respectively. EFL O2 debt was 52 ± 22% greater than control (2.19 ± 0.58 vs. 1.49 ± 0.38 l). We conclude that EFL exercise increases the O2 debt and leads to hypoxemia in part due to hypercapnia.
Introduction
In spite of the fact that dynamic hyperinflation (Calverley and Koulouris 2004; O’Donnell et al. 2001) and peripheral muscle deconditioning (Maltais et al. 1998; Richardson et al. 1999) are important factors limiting exercise in COPD, there is evidence that inadequate oxygen delivery to working locomotor and respiratory muscles may also play an important role (Aliverti and Macklem 2001; Aliverti et al. 2005a; Iandelli et al. 2002).
This situation has been modeled in normal humans by imposing expiratory flow-limitation (EFL) during exercise which limits exercise performance by intense dyspnea (Aliverti et al. 2002; Iandelli et al. 2002), while decreasing arterial oxygen saturation and cardiac output (Aliverti et al. 2005a). This in turn leads to premature competition between respiratory and locomotor muscles for available energy supplies and the early onset of mixed respiratory and metabolic acidosis (Aliverti and Macklem 2001; Kayser et al. 1997).
As a result of these previous experiments (Aliverti and Macklem 2001; Aliverti et al. 2005a; Kayser et al. 1997) we predicted that EFL exercise in normal subjects should increase the oxygen debt by decreasing systemic oxygen delivery to exercising muscles. Accordingly, to test this hypothesis, we measured O2 kinetics during repayment of the O2 debt after control and EFL exercise in young, normal, male subjects. Our results revealed that EFL increased O2 net debt by an average 52% and led to hypoxemia in part due to hypercapnia.
Material and methods
Subjects
We studied six healthy male individuals whose baseline characteristics are shown in Table 1. All subjects gave signed–informed consent and the protocol was approved by the University of Athens Ethics Committee, where the experiments were conducted.Table 1Subjects’ characteristicsSubjectAge (years)Height (m)Weight (kg)FEV1 (l)FEV1 (%predicted)Dyspnea (Borg scale)1381.76783.90971551.752221.73754.231001702.583191.69593.93971001.674221.94865.471121252.555221.88805.731091002.056341.66603.5296901.87Mean261.77734.461021232.16SD80.11110.917330.41FEV1, forced expiratory volume in 1 s in absolute and as percentage predicted; , maximal workload when EFL was imposed during the incremental cycling test;  maximal oxygen uptake when EFL was imposed during the incremental test
Pulmonary function tests
Spirometry for the determination of forced expiratory volume in 1 s (FEV1) was performed by a Vmax 229 system (Sensor Medics, Anaheim, CA, USA) (Table 1).
Exercise protocols
Three exercise tests were performed for each subject on an electromagnetically braked cycle ergometer (Ergoline 800, Sensor Medics) with the subjects maintaining a pedaling frequency of 60 rpm. At the beginning an incremental test was performed to establish the individual subject’s maximal workload when EFL was imposed  by the application of a Starling resistor in the expiratory line limiting flow to approximately 1 l s−1. After 3 min of baseline measurements, followed by 3 min of unloaded pedaling, the work rate was increased every min by 20 W to the limit of tolerance (i.e. the point where the work rate could not be tolerated due to severe sensation of dyspnea, see Table 1). On a separate day, two square-wave protocols were performed: the first without EFL (control) and the second with EFL. After 3 min of baseline measurements, followed by 3 min of unloaded pedaling, the work rate was set and maintained at  for 6 min in both protocols. At the end of exercise, EFL was removed and recovery was monitored for an additional 6 min (Fig. 1).Fig. 1Typical example of volume changes of the chest wall during 3 min of baseline measurements (QB quiet breathing), 3 min of unloaded cycling (W/u warming up), 6 min of exercise at the pre-determined , and 6 min of recovery. Arrows indicate end of expiration and inspiration. Data acquisition was interrupted for 1 min every 2 min in order to allow data storage
Gas exchange and flow measurements
Instantaneous inspired and expired O2 and CO2 concentrations, end-tidal values for O2 and CO2 (PETO2, PETCO2, respectively) and flow at the mouth were recorded breath-by-breath (Vmax 229, Sensor Medics) throughout both control and EFL exercise and recovery runs. Flow displaced at the mouth was also measured by a screen type pneumotachograph (3813 Hans Rudolph, Kansas City, MO, USA) and from these measurements the following parameters were obtained: minute ventilation,  tidal volume, (VT); breathing frequency, (fb); inspiratory (Ti), expiratory (Te), total respiratory (Ttot) cycle time; and duty cycle (Ti/Tot). Cardiac frequency (fc) and percentage arterial oxygen saturation (% SaO2) were determined using the R–R interval from a 12-lead on-line electrocardiogram (Marquette Max, Marquette Hellige GmbH, Germany) and a pulse oximeter (Nonin 8600, Nonin Medical, USA), respectively. The modified Borg Scale (Borg 1982) was used to rate the magnitude of dyspnea and leg discomfort every 2 min throughout exercise.
Operational chest-wall volume measurements
Simultaneously, throughout the square wave exercise and recovery protocols the volume of the chest wall was measured by optoelectronic plethysmography (OEP system, BTS, Milan, Italy) as previously described (Aliverti et al. 1997; Cala et al. 1996; Vogiatzis et al. 2005). In brief, the motion of 89 retro-reflective markers placed front and back over the chest wall from clavicles to pubis was recorded. Each marker was tracked by six video cameras, three in front of the subject and three behind. Subjects grasped handles positioned at mid-sternum level, which lifted the arms away from the rib cage, so that lateral markers could be visualized. Dedicated software, which recognizes the markers on each camera in real time, reconstructed their 3D co-ordinates by stereophotogrammetry and using Gauss’s theorem, calculated inspiratory and expiratory tidal volume variations. Thus, breathing pattern was measured both plethysmographically and by integration of flow. Data acquisition from the OEP system was interrupted for 1 min every 2 min (Fig. 1) in order to allow data storage.
Calculation of breath-by-breath oxygen consumption
The volume of O2 exchanged at the mouth differs from the volume of O2 taken up by pulmonary capillaries if the amount of O2 stored within the lung changes. This occurs if the volume inspired is different from the volume expired in a given respiratory cycle, and/or if alveolar concentrations of O2 change (Capelli et al. 2001). This is particularly important during on- and off-exercise transients (Cautero et al. 2002). The changes in O2 stored within the lung must be subtracted from the volume of O2 exchanged at the mouth in order to obtain the amount of O2 exchanged at the alveolar level. This can be done by combining OEP with independent measures of the subdivisions of lung volume and continuously measuring changes in the absolute gas volume by OEP throughout all exercise tests. This method was recently described in detail (Aliverti et al. 2004a, 2005a). Accordingly, in the present study, breath-by-breath oxygen consumption  was computed throughout using the data acquired by OEP, flow at the mouth and the continuous inspired and expired O2 and CO2 concentration recordings. Using this method corrections were made for (1) pneumotachograph integrator drift, (2) the sampling rates of the O2 and CO2 meters, (3) the time delay of the O2 analyzer and the flow through the O2 and CO2 sampling lines, (4) precise synchronization of O2 concentration and volume signals and (5) any changes in O2 stored within the lung. Thus, in contrast to commercial devices, we measured  at the level of the pulmonary capillaries, not at the mouth.
Curve fitting
To characterize the kinetics of the  response during square-wave exercise and recovery single and double-exponential functions were applied, respectively, to the data using a non-linear least-squares fitting procedure (Figs. 2, 3) without omitting the first 20 s of the response for either the on- or off-transient. The equations applied (Linnarsson 1974; Ozyener et al. 2001) were
where in Eq. 2, the response is the sum of two components, each described by an exponential decay with two time constants: τ1 = 1/b (the fundamental  component) and τ2 = 1/d (the slow  component). In the same equation, a and c are the initial values of these two components and their sum (a + c) represents the initial value of the total response, i.e. the value of  at time zero. The adequacy of the fitting model was tested by calculating R2 values and residuals for single and double exponentials.Fig. 2Typical example of breath-by-breath  during control (closed circles) and EFL exercise (open circles) and recovery. Upon completion of exercise the Starling resistor was removed. Note that time is aligned on the precise instant in which the recovery started (time zero)Fig. 3Typical example of breath-by-breath  during recovery from control (closed circles) and EFL exercise (open circles). Data are fitted with double-exponential curves
Measurement of oxygen debt
Oxygen debt was calculated by integrating, for each subject, the fitting curves between time zero (start of recovery) and an arbitrary time of 400 s (Fig. 3) Then, the value of mean  at baseline, multiplied by the same period of time, was subtracted (Aliverti et al. 2005a).
Estimation of cardiac output and arterio-venous oxygen difference
To estimate the cardiac output (CO) and the arterio-venous oxygen (a-v O2) difference during EFL and control exercise the following equations (Stringer et al. 1997) were used:
Statistical analysis
Data are presented as mean ± SD. Significant differences in recorded variables over the last minute of the square-wave exercise protocols between control and EFL tests were assessed by the Wilcoxon signed-rank test as were recovery data from control and EFL exercise tests. Linear regression analysis was performed using the least-squares method. A statistical significance of 0.05 was used for all analyses.
Results
Square-wave protocols
Figure 2 displays a typical example of breath-by-breath  during control and EFL exercise protocols. Individual and mean values of  sustained during the two square-wave protocols are shown in Table 1, whereas the gas exchange and ventilatory pattern parameters are shown in Table 2.  tended to be lower (by 8 ± 2%) at the end of EFL compared to control exercise, albeit not significantly (Table 2). However, EFL compared to control exercise was associated with significantly lower values for fb, and Ti/Tot. Te at the end of EFL exercise was significantly higher compared to control (Table 2). Over the last minute of EFL exercise, mean values for fc, PETCO2 (54.7 ± 9.9 mmHg) and dyspnea scores were significantly larger compared to control exercise (Fig. 4), whereas mean PETO2 (87.0 ± 11.3 mmHg) was significantly lower (Fig. 4). The mean fall (P = 0.044) in %SaO2 at the end of EFL exercise was 4 ± 3%. (Fig. 4). Leg discomfort at the end of EFL exercise was not different to that at the end of control exercise (Fig. 4). The on-transient data were best fitted with a mono-exponential function where the time constant of the fundamental  component was not significantly different during EFL exercise as compared to control exercise (Table 3).Table 2Exercise data at the end of the 6 min protocols with and without EFL Control exerciseEFL exerciseWork rate (W)111 ± 36109 ± 302.08 ± 0.311.93 ± 0.422.14 ± 0.381.88 ± 0.49*fc (beats min−1	)152 ± 19163 ± 19*59.8 ± 10.238.8 ± 15.1*VT (l)2.08 ± 0.482.05 ± 0.45fb (breaths min−1)29 ± 519 ± 3*Ti (s)1.00 ± 0.100.85 ± 0.15Te (s)1.15 ± 0.232.02 ± 0.43*Ti/Tot (%)46 ± 531 ± 4*Values are means ± SD oxygen uptake;  carbon dioxide output; fc, cardiac frequency;  minute ventilation; VT, tidal volume; fb, breathing frequency; Ti, inspiratory time; Te, expiratory time; Ti/Tot, duty cycle*Significant differences (P < 0.05) between EFL and control exerciseTable 3Individual and mean data on the time constant of the fundamental and slow component of the  response for on- and off-transientsSubjectOn-transient τ1 (s)Off-transient τ1 (s)Off-transient τ2 (s)ControlEFLControlEFLControlEFL175.866.234.729.21,4291027.5250.562.932.123.11,428930.7349.054.633.932.8*976.1489.387.053.430.9*1605.8532.541.236.16.9909855.06m61.772.553.136.61,666727.7Mean59.864.140.626.61358.01020.5SD20.415.69.910.6319.5305.2P value0.2760.0280.068Fundamental (τ1) time constant of the on-transient  response during control and EFL exercise (first and second columns). Fundamental (τ1) and slow (τ2) time constants of the off-transient  response after control and EFL exercise (third to sixth column).*Values > 106Fig. 4Individual values for heart rate (fc, a), %SaO2 (b), PETO2 (c), PETCO2 (d), dyspnea (e) and leg discomfort (f) at the end of control and EFL exercise protocols
Recovery oxygen kinetics and oxygen debt
Figure 3 displays a typical example of breath-by-breath  data fitted with a double-exponential function during recovery from control and EFL exercise square-wave tests. The R2 values of the fitted curves applied to the recovery breath-by-breath  data were not significantly different between EFL (0.89 ± 0.07) and control exercise (0.86 ± 0.09). Immediately after removing the Starling resistor at the end of EFL exercise there was a step increase in  so that repayment of O2 debt started at a higher  after EFL than control. Figure 5 demonstrates individual values of the a, c, and a + c parameters obtained from Eq. 2. All three parameters were significantly higher after EFL compared to control exercise (a: P = 0.04; c: P = 0.0002; a + c: P = 0.02). Also shown in Fig. 5 is the total O2 debt which was 2.19 ± 0.58 l after EFL exercise compared to the control value of 1.49 ± 0.38 l, an increase of 52 ± 22% (P = 0.01) caused by EFL. In order to verify possible problems originated by the curve fitting, oxygen debt and deficit were also calculated directly from the original breath-by-breath data and did not differ significantly compared to the data obtained by the fitted curves (average difference 4.3 ± 3.1%).Fig. 5Individual values of the parameters given in Eq. 1: a (a), c (b), a + c (c) and calculated gross values for the oxygen debt (d) after the EFL and control exercise protocols
Individual and mean values for the off-transient time constants of the fundamental and slow  components are shown in Table 3. The fundamental time constant was significantly (P = 0.028) shorter during recovery from EFL compared to control exercise (Table 3). The off-transient slow component during recovery from EFL tended (P = 0.068) to be shorter compared to that after control exercise; however, in recovery from control exercise the slow component was not evident in subjects 3 and 4 (Table 3). Table 4 reports the individual and mean data of the on and off  transients for EFL and control exercise.Table 4Individual and mean data of the on and off  transientsSubjectOn-transientOff-transientAControl(l min−1)aEFL(l min−1)aControl(l min−1)aEFL(l min−1)cControl(l min−1)cEFL(l min−1)11.9441.4721.912.420.670.7821.7512.0592.334.930.720.9130.9931.0401.852.090.490.6041.8061.7421.872.620.410.6550.9811.7431.403.840.680.9461.5711.4541.291.890.500.67Mean1.5071.5851.782.970.570.76SD0.4210.3470.381.180.120.14P value0.6710.0380.001For the on-transient after control and EFL exercise the  value for a was obtained from Eq. 1:  For the off-transient after control and EFL exercise the  values for a and c were obtained from Eq. 2:
Estimation of cardiac output and arterio-venous oxygen difference
From Eq. 3), COEFL = [1.9 l min−1/5.72 +  (0.105 ×  95
1)] = 12.5 l min−1.
From Eq. 4, a-v O2EFL =  5.72 +  (0.105 ×  95) = 15.2 ml O2 per 100 ml of blood. In addition, a-v O2control = 5.72 +  (0.10 ×  60
2) = 5.72 + 5.9 =  11.7 ml O2 per 100 ml of blood.
Based on the above calculations, the mixed venous blood O2 content (CvO2) for EFL would be equivalent to 4.8 ml per 100 ml blood or 48 ml O2 l−1. For control, CvO2 would be equivalent to 8.3 ml O2 per 100 ml blood or 83 ml O2  l−1.
Taking into account that 70% of blood volume is located in the venous compartment at rest and during moderate exercise that figure falls to approximately 50% (Astrand and Rodahl 1986), then during EFL exercise with a cardiac output of 12.5 l min−1 the CvO2 will be [(12.5 l min−1 ×  0.5) ×  0.048 l O2] =  0.3 l or 300 ml O2. For control exercise, the CvO2 will be [(13.75 l min−1 ×  0.5) ×  0.083 lO2] =  0.57 l or 570 ml O2. Thus, the difference in CvO2 between EFL and control will be 570 − 300 =  270 ml. Hence from the 0.70 l difference in oxygen debt between EFL and control calculated in Table 5 (2.19 − 1.49 =  0.70 l), 0.27 l (i.e. approximately one-third) would account for the replenishment of the blood oxygen stores.Table 5Oxygen deficit and debt for control and EFL exercise Control exerciseEFL exerciseO2 deficit (ml)1,448 ±  7391,692 ± 575O2 debtfast component (ml)1,174 ± 2741,232 ± 470O2 debtslow component (ml)3,470 ± 5514,201 ± 683*O2 debttotal (ml)4,645 ± 4785,433 ± 763* (ml)3,157 ± 4433,238 ± 539O2 debtnet (ml)1,487 ± 3842,195 ± 581*Values are means ± SD oxygen uptake*Significant differences (P < 0.01) between EFL and control exercise
Discussion
There is growing evidence that an inadequate oxygen supply to meet demand may play a more dominant role in limiting exercise capacity in some patients with advanced COPD than impaired lung function (Aliverti et al. 2004b, 2005b; Maltais et al. 1998; Oelberg et al. 1998; Potter et al. 1971; Richardson et al. 1999; Stark-Leyva et al. 2004). In healthy subjects, limiting expiratory flow by a Starling resistor or increasing expiratory load by imposing a constant pressure at the mouth during expiration, have been shown to significantly reduce cardiac output during exercise (Aliverti et al. 2005a; Stark-Leyva et al. 2004). This results from the enforced decrease in the velocity of shortening of expiratory muscles and hypercapnia, both of which contribute to increased expiratory muscle force and decreased duty cycle so that expiration acts like a Valsalva maneuver with inadequate time to recover during inspiration (Aliverti et al. 2002, 2005a; Iandelli et al. 2002). Furthermore, the decreased duty cycle magnifies the effect of high expiratory pressures when averaged over the whole respiratory cycle. Our present results show that EFL increased O2 debt by an average 52% and led to hypoxemia in part due to hypercapnia. This supports our hypothesis that the reduction in cardiac output during EFL exercise decreases O2 supply to working locomotor and respiratory muscles resulting in an increased O2 debt. We conclude that the decreased cardiac output is important in limiting EFL exercise performance in healthy subjects.
EFL as a model for COPD
The experimental model used in this study was designed to simulate, in part, the flow-limitation commonly experienced by COPD patients during exercise. The pros and cons of this model have been previously extensively discussed (Aliverti et al. 2002; Iandelli et al. 2002). The model has demonstrated that exercise with EFL induces intolerable dyspnea, CO2 retention, impaired exercise performance, expiratory muscle recruitment (Kayser et al. 1997), blood shifts from trunk extremities, a reduced duty cycle, arterial desaturation, and a decrease in cardiac output (Aliverti et al. 2005a).
In the present study, we confirmed many of these results and showed that the increased O2 debt was accompanied by a reduction in  by 8% and a fall in SaO2 by 4% at the end of EFL exercise, thus leading to a reduction in systemic O2 delivery of ∼  12%, in close agreement with the 15% reduction reported by Aliverti et al. (2005a). It is reasonable to assume that the reduction in systemic O2 delivery during EFL exercise would be associated with the progressive recruitment of fast-twitch fibers and hence the premature onset of the lactate threshold. The latter has been shown to be the case during exercise with EFL (Aliverti et al. 2005a). When exercise workloads exceed the lactate threshold, energy supplies are inadequate to meet demands. The resulting competition between working locomotor and respiratory muscles for the available energy supplies, regulated by autonomic reflex mechanisms (Harms et al. 1998), would be substantially worsened by the decrease in the available O2 and should further increase the lactate production. It is therefore likely that the muscle and blood lactate levels (not measured in the present study) would be appreciably higher during EFL exercise and consequently, the lactate related metabolic cost could significantly contribute to the repayment of the O2 debt in recovery from EFL exercise.
In addition to the reduction in systemic O2 delivery, the present study shows that application of EFL during exercise significantly decreased minute ventilation compared to control exercise, causing hypercapnia. This confirms earlier results that show that a vicious circle is induced whereby increasing central ventilatory drive increases expiratory pressure which further reduces alveolar ventilation and cardiac output (Aliverti et al. 2002, 2005a; Iandelli et al. 2002). Furthermore, the observed elevated heart rate during EFL exercise possibly reflects a reduction in stroke volume secondary to the decrease in venous return. In the study by Stark-Leyva et al. (2004), expiratory loading during exercise increased heart rate in an attempt to minimize the effects of the reduced stroke volume on cardiac output. Thus, the reduced cardiac output secondary to the high expiratory pressures would also be expected to contribute importantly to the greater oxygen debt that was measured after EFL exercise.
In summary, EFL exercise in healthy subjects reproduces many features of COPD including acute respiratory failure, a condition resembling cor pulmonale, dyspnea, and impairment of exercise performance. Furthermore, it has led to testable predictions, one of which is the rationale for this study and which to date have been proven to be accurate (Aliverti et al. 2004b, 2005a, b).
Off-transient O2 kinetics
The increase in O2 debt by 52% that we found during EFL exercise impacted, as predicted, on O2 kinetics during recovery. Accordingly, the repayment of O2 debt after EFL exercise started at a higher  compared to control and the off-transient time constant of the fundamental and slow  components were shorter after EFL exercise. Collectively, the findings describing the EFL off-transient O2 kinetics reflect a more rapid replenishment of blood O2 stores (fundamental component) and a faster repayment of O2 tissue debt (slow component), both of which could result from the greater O2 deficit during EFL exercise as opposed to control exercise. Indeed, if we considered that cardiac output during EFL exercise was lower than control and  was not significantly different between EFL and control, then it would be reasonable to expect that during exercise with EFL, that is known to decrease pulmonary blood flow (Aliverti et al. 2005a), the arterial to mixed venous blood O2 difference would be larger and the mixed venous blood O2 content would be lower. This notion was confirmed by our calculations of arterial to mixed venous blood O2. When at the end of exercise EFL was removed, the sudden increase in cardiac output and in blood perfusing the lungs brought about a refilling of the O2 stores of mixed venous blood; hence the sudden increase of  (marking the replenishment of the body’s deprived oxygen stores), a faster  kinetics and a larger O2 debt in EFL (Table 5). The larger depletion of the inner oxygen stores is also suggested by the longer time constants of the on- and off-transient  response during EFL exercise (Table 3). Our calculations showed that approximately 30% of the O2 debt difference between EFL and control exercise was due to the blood O2 replenishment (Table 5) caused by the reduced cardiac output during EFL, whereas the rest was due to other possible mechanisms described below.
In line with the results of Cunningham et al. (2000) and Ozyener et al. (2001) describing O2 kinetics after heavy exercise, the off-transient  data after EFL exercise were best fitted by a two component exponential function. The fundamental off-transient component after control exercise (40.6 s) was similar to that (∼  33 s) described by Cunningham et al. (2000) and Ozyener et al. (2001) following very heavy exercise, lasting as in the present study for 6 min. On the other hand, the time constant of the fundamental component after EFL exercise (26.6 s) was significantly shorter than that of control exercise, thus confirming replenishment of the body’s deprived oxygen stores upon removal of EFL. In addition, the shorter time constant calculated after EFL is the result of fitting the data from higher starting points as evidenced by the significantly higher a + c values shown in Table 4 and Fig. 5c.
Furthermore, the time constant of the slow component after EFL exercise (1,020 s) was twofold longer than the one (460 s) described previously for very heavy exercise (Ozyener et al. 2001) possibly reflecting the additive effects of the EFL-induced reduction in systemic O2 delivery on the repayment of tissue O2 debt. Although at present the mechanism(s) of the  slow component is not fully understood, there are important factors that could influence the slow component after EFL exercise. These include the blood lactate concentration (Poole et al. 1994), the influence of the metabolic acidosis on the HbO2 dissociation curve (Wasserman et al. 1991), the increased respiratory and cardiac muscle energy requirement associated with EFL exercise (Aaron et al. 1992; Harms et al. 1998), the progressive recruitment of type-II fibers (Coyle et al. 1992) and to a lesser extent the increased levels of circulating catecholamines (Gaesser et al. 1994) associated with the greater cardiovascular response during EFL exercise. Importantly, excessive expiratory muscle recruitment has been shown in patients with airflow limitation to increase the oxygen cost of breathing threefold (Aliverti et al. 2004b). Accordingly, it is reasonable to assume that the higher O2 cost of breathing during EFL exercise would significantly enhance the O2 dept as compared to exercise without EFL.
In the present study, we utilized a double-exponential function to describe the off-transient  kinetics not only for EFL exercise but also for control exercise [typically fitted by a mono-exponential function (Cunningham et al. 2000; Ozyener et al. 2001)], in order to allow adequate comparisons of relevant parameters of recovery for both exercise tests. Accordingly, the slow component observed after control exercise was not discernible in two subjects using the double-exponential function. This is in accordance with previously reported data by Cunnnigham et al. (2000) who exercised healthy subjects at a similar work rate (100 W), yielding similar exercise  (1.7 l min−1) as the one reached in the present study during control exercise. In conditions where exercise is sustained at a moderate intensity, as in the control test, the off-transient slow component is often not discernible such that  kinetics can retain first-order characteristics (Cunnnigham et al. 2000; Gerbino et al. 1996; Ozyener et al. 2001).
In conclusion, the results of the present study provide further evidence that expiratory flow limitation during exercise reduces systemic O2 delivery, enhances the O2 debt and leads to hypoxemia in part due to hypercapnia.

oxygen debt
exercise tolerance
copd models


Calcaneal nonunion: three cases and a review of the literature

The long-term follow-up of intra-articular calcaneal fractures is often accompanied by complications. Frequently occurring are arthrosis, arthrofibrosis of the subtalar joint, and malunion. Uncommon is the calcaneal nonunion. A total of three cases is presented in this report, including a review of the literature. The occurrence of a nonunion appears to be more common after conservative treatment, but the pathophysiology remains unclear, however smoking may play a role.
Introduction
During long-term follow-up after intra-articular calcaneal fractures complications frequently occur. In clinical practice, arthrosis and arthrofibrosis of the subtalar joint, malunion and nonunion are encountered [8, 12]. Nonunion is only rarely found, and studies concerning complications after intra-articular calcaneal fractures do not describe this complication [5, 7]. In only five studies, including nine patients, reports on nonunion are made [3, 6, 10, 11, 13]. Three patients with a pseudarthrosis after an intra-articular fracture of the calcaneus are presented in the current study.
Cases
Case 1
A male adult (age 49) fell from a height of 2 m fracturing his left calcaneus. According to the Essex–Lopresti classification he sustained a joint-depression type, and a type IIA fracture according to the Sanders classification. There was no injury to the soft tissues. After 6 days the fracture was operated on, using percutaneous reduction and fixation according to the technique of Forgon and Zadravecz [9]. Post-operative treatment consisted of active range of motion exercises and 3 months non-weight bearing. Initial recovery was uncomplicated. Two years after the trauma, the patient however returned to our outpatient department with complaints of painful walking, and unable to work. A computed tomography (CT) scan showed a pseudarthrosis of the calcaneus and talocalcaneal joint surface incongruence (Fig. 1). An injection of 10 cc lidocain in the subtalar joint temporarily reduced pain. A subtalar joint arthrodesis was performed, fusing the talus and calcaneus with the use of bone graft gained from the iliac crest. After 1 year the arthrodesis has fully consolidated, the patient is pain free, but has currently not yet returned to work.
Fig. 1a Case 1: Axial CT-scan image 22 months after percutaneous reduction and fixation showing nonunion; b Sagittal view
Case 2
A female patient, aged 53, sustained a fracture of the right calcaneus after a fall from the stairs (Essex–Lopresti joint depression type, Sanders type IIB). As patient was seen three weeks after the trauma, the initial treatment was conservatively, consisting of 1 week of plaster-of-Paris and 3 months non-weight bearing with active range of motion exercises. Two months later the patient was unable to bear weight at the fractured side due to pain. A CT showed a delayed union of the calcaneus (Fig. 2). Peroperatively, 6 months after trauma, there was a nonunion of the fracture, without apparent signs of arthrosis of the talocalcaneal joint surface. Because of the smoking habits and diabetes in this patient, as independent causes of high wound complication rates, an early subtalar arthrodesis with bone graft harvested from the tibial tuberosity was performed instead of a correcting osteotomy. Three months after the salvage operation the arthrodesis had consolidated, the patient is walking pain free.
Fig. 2a Case 2: Semi-coronal CT-scan image 5 months after conservative treatment showing delayed union; b Sagittal view
Case 3
A male adult, of 39-years-old, sustained a bilateral calcaneal fracture after jumping of a first storey balcony. The radiographs showed a comminuted intra-articular calcaneal fracture according to the Essex–Lopresti classification at both sides and Sanders type IIC and IIIAC fractures on the left and right foot, respectively. Initial treatment was conservative, consisting of 1 week of plaster-of-Paris and 3 months non-weight bearing with active range of motion exercises. Thirteen months later the patient returned to the outpatient department with complaints of pain at the left heel. Additional radiographs and CT showed a nonunion of the fracture of the left calcaneus (Fig. 3). A subtalar arthrodesis was performed, with bone graft from the tibial tuberosity. Two cannulated screws were inserted from the tuberosity of the calcaneus, bridging the primary fracture line. The after treatment was complicated with a superficial infection, which could be treated adequately with intravenous antibiotics. Six months after the secondary fusion the patient was able to walk pain free.
Fig. 3a Case 3: Semi-coronal CT-scan image 13 months after conservative treatment showing nonunion; b Sagittal view
Discussion
Improper healing after an intra-articular calcaneal fracture carries a high morbidity. Three groups: A, malunion; B, nonunion; and C, osteonecrosis, were suggested by Zwipp [13]. These groups are further divided according to the presence of joint incongruence, varus/valgus, loss of height, translation of bones and luxation. Treatment of these complications after intra-articular calcaneal fractures depends upon the presence of these conditions [13].
The first written report on calcaneal fracture nonunion was by Thomas (1993). He presented a 36-year-old female patient, treated non-operatively, who developed a calcaneal nonunion after 6 months [11]. A correction of the displaced fragment was performed and stabilized using plate osteosynthesis and bone graft. The patient was able to fully bear weight 12 weeks after this procedure.
Gehr described a 38-year-old male patient with an intra-articular, comminuted fracture of the calcaneus [3]. This fracture was treated with open reduction and internal plate osteosynthesis. After removal of the plate at 18 months the patient returned to the clinic with local swelling and pain during walking. A nonunion was seen and a correction osteotomy was performed with bone graft and screw fixation. The fracture showed healing at 8 weeks.
Karakurt et al. [6] presented one 42-year-old male patient with a nonunion of the calcaneus after conservative treatment. The patient sustained a comminuted, open calcaneal fracture which was treated in plastercast for 6 months. Eight months after trauma the patient was unable to walk without crutches because of severe pain of the heel. After removing fibrotic tissue the calcaneus was filled with bone graft. Eight months after the operation patient was able to walk and work without pain.
Zwipp and Rammelt [13] reported two patients with a nonunion of the calcaneus. A 61-year-old female patient, with a Sanders IIC fracture with a luxation of the tuberosity fragment, was treated conservatively. An arthrodesis of the subtalar and calcaneocuboid joint was performed 6 months after trauma. Postoperatively the American Orthopaedic foot and Ankle Society hindfoot score improved significantly, compared with pre-operative values. The second patient was a 45-year-old female polytrauma patient. After percutaneous reduction and fixation she was diagnosed with a nonunion 1 year after the trauma, for which a subtalar joint arthrodesis was performed. No data on outcome was provided in this case.
From cases described in literature and in the current report no similarities that could indicate a risk factor of nonunion could be found. There are no apparent similarities besides the nonunion (Table 1). There might be a trend of nonunion occurring after conservative treatment, suggesting that less rigid or no fixation may play a role in causing fracture nonunion in the calcaneus. However Howard et al. [5] saw no cases of nonunion in a group of 164 intra-articular calcaneal fractures treated nonoperative. Patient age varied between 36 and 61, both sexes were affected equally often and initial treatment differed between patients. Karakurt et al. [6] suggested that smoking could be the cause of the nonunion. All patients in this report were smokers. For tibial fractures strong evidence exists for delayed fracture healing in smokers [4]. And a significant lower union-rate was seen after subtalar arthrodesis in smokers versus non-smokers [2]. Assous et al. [1] however saw no difference in fracture healing between smokers and non-smokers in a small series of intra-articular calcaneal fractures treated operatively.
Table 1Summary of patients presented in the literature after a calcaneal nonunionStudyGenderAgeInitial treatmentSalvage procedureFollow-up*(months)Thomas and Wilson [11]Female36ConservativeOsteotomy, plate, bone graft3Thermann et al. [10]–––Subtalar arthrodesis62Gehr et al. [3]Male38ORIFOsteotomy, screws, bone graft2Karakurt et al. [6]Male42ConservativeBone graft8Zwipp and Rammelt [12]Female61ConservativeSubtalar arthrodesis, calcaneocuboid joint fusion–Female45PercutaneousSubtalar arthrodesis–Current studyMale49PercutaneousSubtalar arthrodesis14Female53ConservativeSubtalar arthrodesis3Male39ConservativeSubtalar arthrodesis6* The follow-up period after the salvage procedure is given
In contrast to the infrequent occurrence of the calcaneal nonunion, stated in the case reports above, Thermann et al. [10] describe an in incidence of 10% nonunion (n = 4/40) in a group of patients receiving a subtalar arthrodesis for persisting invalidating pain after an intra-articular calcaneal fracture. In these four patients the nonunion coincided with painful subtalar posttraumatic arthrosis, delineating the indication for a subtalar arthrodesis, instead of a correction osteotomy with internal fixation.
Calcaneal nonunion has been reported on infrequently in the literature, but the number of patients seen by Thermann, and our own series, suggests that the incidence might be higher than expected.

nonunion fracture pseudarthrosis calcaneus


Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant.
Introduction
Cytochromes P450 (CYPs) are enzymes responsible for the oxidative biotransformation of a large variety of endogenous and exogenous substrates, like steroids, carcinogens and drugs, and have been found in virtually all organisms [1]. In human drug metabolism, one of the most important CYP isoforms is CYP2D6. This hepatic enzyme is involved in the phase I oxidative metabolism of about 30% of currently marketed drugs, including much prescribed antidepressants, β-blockers, opioids and antiarythmics [2]. The enzyme is known for its genetic polymorphisms and gene multiplicities, even increasing its clinical relevance [3]. Structural studies on CYP2D6 are of crucial importance to understand how this enzyme works and to be able to predict the metabolism of new (druglike) compounds [4].
A common structural feature in all CYPs, including CYP2D6 [5], is the distal helix (I-helix) positioned above the heme plane, which constitutes an important part of the enzyme’s active site (Fig. 1a). Previously, we demonstrated the critical role of the I-helix residue T309 in the mechanism of oxidation by CYP2D6 [6]. Similar results were found after mutation of the homologous threonine residues T302 and T303 in rabbit CYP2B4 and CYP2E1, respectively [7, 8]. The I-helix threonine is highly conserved in CYPs and seems to be a key residue in the monooxygenation reaction. It has been studied in many CYPs with mutagenesis, spectroscopic and crystallographic techniques [9]: depending on the specific isoenzyme, the distal threonine has been attributed a variety of roles besides oxygen activation and proton delivery [9, 10], including electron transfer [11], substrate recognition [12] and control of heme spin state equilibrium [12–17]. This multiplicity of roles, as indicated by the diversity of results obtained from different CYPs, has been interpreted as a lack of a universal role of this residue, which might have instead distinct functions within the CYP superfamily [9, 14, 17]. Fig. 1a The cytochrome P450 2D6 (CYP2D6) active site as obtained from crystallographic data (PDB 2F9Q). The heme (coordinated by C443) and part of the I-helix are shown in color with the heme and the residues A305 and T309 as ball-and-stick models. Hydrogen atoms are not modeled in the PDB file. b Optimized geometry of the CYP2D6 active site model, i.e., the A305–G306–M307–V308–T309 part of the I-helix and the iron porphyrin part of the heme catalytic center of CYP2D6 in its doublet (low-spin) electronic spin state, with a water molecule coordinated to the iron (position 1). c Geometry of the CYP2D6 active-site model corresponding to a second energy minimum (water in position 2). Carbon, nitrogen, oxygen and hydrogen atoms are colored green, blue, red and white, respectively. Iron and sulfur atoms are colored orange. Distances are in angstroms
In this study, spectroscopy is used to probe the active site of the wild type and the T309V mutant of CYP2D6 to establish whether the mutation influences the heme characteristics. Resonance Raman (RR), a vibrational spectroscopy technique based on inelastic scattering of laser radiation in resonance with an electronic transition of a chromophore, is used to obtain information about several properties of the heme prosthetic group [18, 19]. The heme-iron oxidation state, spin state and coordination state, as well as conformational changes of the vinyl and propionate side chains of the porphyrin macrocycle can be inferred from RR spectra [19]. Furthermore, the Fe–CO stretching frequency of the ferrous CO–heme complex is sensitive to variations in the electrostatic potential of the heme distal pocket induced by substrate-binding or mutated residues [20, 21]. RR was successfully employed in the past to study many CYPs [19] and the effects of mutations in their active site [22–27], including those of the conserved I-helix threonine [11, 28, 29].
Besides spectroscopic data, a computational approach is employed to rationalize the experimental results. Although experimental data on threonine mutants are already available for many CYPs, theoretical descriptions have been only carried out at a classical force-field level for the T302A mutant of CYP2B4 [30] and at a density functional theory (DFT) level for the T252A mutant of CYP101 [31]. Quantum-mechanical DFT calculations can reliably predict the relative energies of the various electronic states of iron(II) and iron(III) porphyrins and have been previously used to study the resting state as well as further steps of the catalytic cycle of CYPs [31–35]. Therefore, this method, in absence of decisive experimental proof in favor of a specific model, is used in the present work to test the compatibility of a definite hypothesis with available structural and spectroscopic data on the CYP2D6 active site. In particular, through a computational analysis of the wild-type and the T309V-mutant active-site structures, interactions are investigated between residue T309, the heme moiety and a water molecule located above the heme in the resting state.
Materials and methods
Materials
The pSP19T7LT plasmid containing in tandem the complementary DNAs (cDNAs) of human CYP2D6 with a C-terminal His6-tag and the human NADPH-CYP reductase, was used as described before [6]. Escherichia coli JM109 was obtained from DSMZ (Braunschweig, Germany). Dextromethorphan hydrobromide (DX) was obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and were obtained from standard suppliers.
Expression and purification of the enzymes
The pSP19T7LT plasmids containing the wild type or the T309V mutant of CYP2D6 cDNA were transformed into E. coli strain JM109. Expression and membrane isolation was carried out as described in [6]. Membranes were resuspended in 0.5% of the original culture volume of potassium phosphate–glycerol buffer (50 mM potassium phosphate buffer, pH 7.4, 10% glycerol) and the enzymes were purified, using nickel affinity chromatography as described in [6]. CYP concentrations were determined by CO difference absorption spectra according to the procedure of Omura and Sato [36], with a Pharmacia Ultrospec 2000 spectrometer.
RR spectroscopy
Spectroscopic measurements were conducted using a homebuilt Raman microscope in a backscattering configuration. A Zeiss microscope (D-7082 with ×40 objective, numerical aperture 0.60, working distance 2 mm) was coupled to a single monochromator (Instruments, Metuchen, NJ, USA) with a mounted grating with 2,400 grooves per millimeter and a CCD camera (DV-420OE, Andor Technologies, Belfast, UK). The 413.1-nm line of a continuous-wave Kr ion laser (Coherent Innova 300c, Coherent, Santa Clara, CA, USA) and the 457.9-nm line of a continuous-wave Ar ion laser (Spectra Physics 2000–336, Newport Corp., Mountain View, CA, USA) were used for excitation, and the Rayleigh scattered light was removed using third Millennium edge long pass filters (Omega Optical, Bratleboro, VT, USA). Laser powers of 5 mW (at 413.1 nm) and 1 mW (at 457.9 nm) at the sample were used throughout the experiments. The sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software. The fitting of experimental data with Lorentzian functions was performed with PeakFit 4.12 (SeaSolve Software, Richmond, CA, USA). For RR measurements, a 1-mm-diameter glass capillary was filled with 10 μl of various concentrations (1–50 μM) of CYP2D6 in potassium phosphate–glycerol buffer and put in a spinning capillary holder under the microscope objective. To measure substrate binding, substrate concentrations of 5–10 mM were used, leading to a 500–1,000-fold excess of substrate with respect to the enzyme (saturating conditions, as indicated by the Ks reported for the substrates [6]). No further spectral changes were detected upon increasing substrate concentration, indicating that all the CYP2D6 in the sample was bound to the substrate [37]. To obtain the ferrous CO complexes, oxygen was removed from the CYP2D6 samples (with or without substrate) by stirring under a nitrogen atmosphere; then the samples were reduced by adding 5 μl of 25 g l−1 sodium dithionite (in potassium phosphate–glycerol buffer) to 20 μl of enzyme in the same buffer, and were exposed to CO for 2–3 min while stirring. During all measurements, the capillary containing the sample was kept spinning to minimize local heating and photodissociation of the iron-bound CO. Before and after every RR measurement, CYP integrity was monitored by CO difference absorption spectroscopy.
Computational details
Model
The crystal structure at 3.0-Å resolution of the substrate-free human CYP2D6 (PDB 2F9Q) [5] was used as the starting structure for the DFT calculations. The computational model includes only a small part of the distal I-helix from A305 to T309, the heme moiety and the proximal C443. In this model, the isopropyl amino acid side chain of V308 is replaced by a methyl substituent and the 2-(methylthio)ethyl side chain of M307 is replaced by a hydrogen. These alterations are applied to considerably reduce the computational cost and are usually justified by the fact that these amino acid side chains are not pointing toward the heme cavity [38]. Likewise, the heme substituents are replaced by hydrogens, and the C443 proximal heme ligand is mimicked by a thiolate anion (HS−). Moreover, a water molecule is added above the heme iron to generate a six-coordinated (6c) resting state, as suggested by spectroscopic data (see “Results” and “Discussion”). This results in an 89-atom model for the active site of the wild-type protein. The T309V mutant is approximated by replacing the alcohol moiety of the T309 by a methyl substituent to give V309 instead. These two models are used to provide a rationale for the differences between the observed relative amounts of high spin (HS) and low spin (LS) in the wild-type and T309V-mutant enzymes of CYP2D6.
Method
DFT calculations were performed with the Amsterdam Density Functional program [39–41]. The atomic orbitals on the atoms of the alcohol moiety of T309, the carbonyl group of A305, the thiolate anion, iron and nitrogens of the heme moiety were described by an uncontracted triple zeta valence plus polarization (TZP) Slater-type orbital (STO) basis set. A double zeta valence plus polarization (DZP) STO basis set was used for the other atoms of the I-helix and the atoms of the periphery of the heme. The inner cores of carbon, nitrogen, and oxygen (1s2) and those of sulfur and iron (1s22s22p6) were kept frozen. The exchange–correlation potential was based on the newly developed generalized gradient approximation exchange functional OPTX in combination with the nonempirical PBE correlation functional (OPBE) [42, 43]. The OPBE density functional was chosen because of its demonstrated superior performance in describing the spin states and electronic structures for iron complexes, which is of crucial importance in the present study [32]. The model was based on the geometry of 2F9Q [5] and is fully optimized for the doublet spin state (LS). This structure was used as a starting structure for the linear transit calculations (geometry scan) in which the water molecule was moved toward the threonine by decreasing the Owater–OThr distance to yield the five-coordinated (5c) heme moiety. In these geometry optimizations, the positions of the α carbons of A305 and T309 and two porphyrin meso carbons were fixed to mimic the rigidity of the protein tertiary structure (see supplementary material). Subsequently, analogous geometry optimizations of the LS geometries obtained were performed for the sextet spin states (HS).
The energy profiles for the mutant enzyme model were obtained by the same procedure for each structure of the wild-type profile in which the threonine hydroxyl moiety was replaced by a methyl substituent.
Results
Enzymes expression and purification, UV–vis absorption spectroscopy
Recombinant expression followed by affinity chromatography purification yielded approximately 60 nmol CYP2D6 per liter of cultured E. coli, in the case of the wild type and the T309V mutant of CYP2D6. The enzymes were pure, as judged by the observation of a single band of 55 kD on a Coomassie brilliant blue stained sodium dodecyl sulfate–polyacrylamide gel. The absolute absorbance spectra of oxidized and reduced CO-bound, wild-type and T309V-mutant CYP2D6 are shown in Fig. 2. Both enzymes show characteristic strong absorption bands at 418 and 448 nm, in their resting state and reduced CO-bound form, respectively. No significant differences are observed in the position or the relative intensity of absorption bands between the spectra of the wild type and the T309V mutant of CYP2D6, even by use of spectral subtraction (data not shown). An amount of P420 (inactive CYP, observed as a band at 420 nm in the reduced CO-bound form) was present in the both the wild-type and T309V-mutant samples, to approximately the same extent. Fig. 2Electronic absorption spectra of 1 μM purified wild-type and T309V-mutant CYP2D6, in the oxidized resting state form (solid line) and the reduced CO-bound form (dashed line)
RR spectroscopy
The RR spectra of the wild type and the T309V mutant of CYP2D6 in the resting state (i.e., without substrate) are at first sight rather similar (Fig. 3). In both cases, the most intense band at 1,372 cm−1 is attributed to the ν4 vibrational mode, indicative for an oxidized heme iron atom [19]. Fig. 3Resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6. Spectra were acquired as described in “Methods.” Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s
The high-frequency region of the RR spectrum of the wild-type enzyme (Fig. 4) is indicative for a predominantly 6cLS oxidized heme, in agreement with a previous study [27]. The bands at 1,501, 1,582 and 1,635 cm−1 are attributed to the ν3, ν2 and ν10 vibrational modes for a 6cLS heme, respectively [19]. However, the slight asymmetry of ν3 suggests the presence of another band at lower wavenumbers, characteristic of a 5cHS species. A better fit of the spectroscopic data for wild-type CYP2D6 is obtained when including bands at 1,486, 1,567 and 1,623 cm−1 for, respectively, the ν3, ν2 and ν10 vibrational modes of a 5cHS heme species in addition to the 6cLS bands. Fig. 4Spin marker band region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6, together with their difference spectrum. Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s. The Lorentzian functions used to fit the spectrum of the wild-type enzyme (see “Methods”) are represented as solid lines (for high-spin marker bands) or dotted lines (low-spin marker bands). Frequencies of high-spin marker bands are reported in bold
The presence of 5cHS heme species in the RR spectrum of the wild type is more evident when compared with the spectrum of the T309V mutant (Fig. 4). Although the two spectra seem similar, the difference spectrum of the two enzymes shows an intensity increase for the bands characteristic of a 6cLS species and a concomitant decrease of the ν3, ν2 and ν10 vibrational modes that are typical for a 5cHS heme. Therefore, in the T309V mutant the content of 5cHS is decreased compared with that in the wild type, with a concomitant increase of the 6cLS fraction.
In the presence of saturating amounts of the substrate DX, the bands distinctive of the 5cHS species (ν3 and ν2 at 1,486 and 1,567 cm−1, respectively) increase in intensity, whereas the intensity of the corresponding bands for the 6cLS species (ν3 and ν2 at 1,501 and 1,582 cm−1, respectively) diminishes for both the wild type and the T309V mutant of CYP2D6 (Fig. 5). The intensity ratio between the 6cLS and 5cHS ν3 bands (at 1,501 and 1,486 cm−1, respectively) is higher in the T309V mutant than in the wild type for substrate-bound CYP2D6. Fig. 5Spin marker bands region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6 in the presence (solid lines) and in the absence (dotted lines) of the substrate dextromethorphan hydrobromide. The spectral difference between the enzyme in the presence and in the absence of substrate is shown for the spin marker band ν3. Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s
The T309V mutation does not alter the Fe–CO stretching frequency (νFe–CO) observed at 476 cm−1 in both the reduced CO-bound wild-type and the T309V-mutant CYP2D6 (Fig. 6). As previously observed [27], in the RR spectrum of wild-type CYP2D6 νFe–CO increases by 5 cm−1 upon addition of saturating amounts of DX. In the same experimental conditions, the νFe–CO band of the T309V mutant broadens toward higher wavenumbers, indicating that a shift, although less evident, is taking place similarly to that in the wild type. Fig. 6Low-frequency resonance Raman spectra of the reduced CO-bound CYP2D6 wild type (WT) and mutant (T309V), in presence and in absence of dextromethorphan hydrobromide (DX). Excitation 457.9 nm, laser power at the sample 1 mW, accumulation time 600 s
DFT calculations
The optimized geometry of the enzyme’s resting state was modeled by adding a water molecule above the heme using the crystal structure of the human CYP2D6 (Fig. 1b). The root mean square deviation of the optimized backbone of the I-helix and the corresponding residues in the experimental structure is only 0.28 Å, confirming that the modifications applied to the side chains of residues M307 and V308 in the model do influence the conformation of the I-helix in the active site. The water molecule is bound to the iron (water position 1 in Fig. 1b, Fe–O distance is approximately 2.2 Å), yielding a 6cLS iron porphyrin complex that is energetically favored over the 6cHS state by 7.5 kcal mol−1. The water molecule is hydrogen-bonded to the carbonyl oxygen atom of the A305-G306 peptide bond (approximately 1.9 Å). The hydroxyl moiety of the threonine amino acid residue (T309) also forms a weak hydrogen bond to this amide oxygen (approximately 2.6 Å). When the water molecule migrates from the iron to the threonine moiety, a hydrogen bond is formed (Fig. 1c) in which the threonine alcohol group acts as a hydrogen-bond donor; the conformation with the OH group acting as a hydrogen-bond acceptor is 6.6 kcal mol−1 less stable.
The energy profiles upon migration of the water molecule toward the threonine (i.e., from position 1 to 2) for both the LS and the HS states are shown as solid curves in Fig. 7. This movement of the water molecule results in the increase of the relative energy of the LS state and a new local minimum (5cLS) is obtained, which is 1.4 kcal mol−1 higher in energy with respect to the energy of the starting structure (6cLS). The relative energy of the HS state increases slightly upon shortening the threonine–water distance and decreases again to reach a new minimum that is lower in energy. There is a spin crossover at a Owater–OThr distance of approximately 3.1 Å and a global minimum for the HS state is obtained. In the corresponding structure, the water molecule is hydrogen-bonded to the T309 and occupies a second position in the active-site pocket, further referred to in the following sections as position 2 (Fig. 1c). Fig. 7The relative doublet (low spin, filled circles) and sextet (high spin, filled squares) energies for the active-site model of the wild-type CYP2D6 with respect to the distance between the oxygen atoms of the water molecule and the threonine alcohol side group. The dashed line represents the low-spin (open circles) and high-spin (open squares) energy profiles for the mutant model, obtained by replacing threonine with valine. LS low spin, HS high spin, 5c five-coordinated, 6c six-coordinated
Replacement of the threonine with valine in each of the structures followed by partial geometry optimizations results in different energy profiles for the mutant (dashed curves in Fig. 7). For both the HS and the LS mutant models, after a small initial decrease of approximately 2 kcal mol−1 which yields minima at an Owater–OThr distance of 3.9 and 3.5 Å for the LS and HS states, respectively, the relative energy increases steeply as the water approaches position 2, for which, in contrast to the wild type, no minima are found.
The interaction between residue T309 and the CO adduct was studied by full geometry optimization of the CO-bound iron porphyrin model species for both the wild type and the T309V mutant, showing that the bond distances, bond dissociation energies and bond orders of the Fe–C≡O moiety are virtually identical (Table 1). Population analysis of the CO adduct shows a substantial iron backdonation into the empty π* orbitals of CO, with occupation numbers of approximately 0.2 electrons for both the wild type and the mutant. Table 1Selected bond distances, bond dissociation energies [ΔE(Fe–CO)], bond orders and π*(CO) orbital population of the CO–heme model for the wild type and the T309V mutant of cytochrome P450 2D6Fe–COFe–C≡OWild typeMutantWild typeMutantDistance (Å)1.8241.8511.1471.146ΔE(Fe–CO) (kcal mol−1)−24.6−24.2––Mayer bond order0.210.200.520.52π*(CO) population––0.19 e−0.18 e−
The superimposed geometries of the fully optimized and experimental structures, of the partially optimized structures of the T309V mutant with water in positions 1 and 2, and of the fully optimized structures of the CO-bound species for both the wild type and the T309V mutant are available as supplementary material.
Discussion
In the resting state, the CYP heme iron atom is generally observed as oxidized (Fe3+), and mainly in the 6cLS state with a water molecule (or hydroxyl ion) as the sixth distal ligand trans to the endogenous cysteinate ligand [44]. Although no distal water ligand is reported in the structure determined by X-ray diffraction [5] (Fig. 1a), both electronic absorption and RR spectroscopy indicate that wild-type CYP2D6 is predominantly 6cLS. The present DFT calculations show that the resting state is clearly a 6cLS state, in agreement with spectroscopic data (Fig. 1b). In addition to experimental data, DFT calculations suggest a network of hydrogen bonds between the residue A305, the distal water ligand and the residue T309, in which T309 and the water ligand are too far to interact directly. The same structural pattern, where the distal water is hydrogen-bonded to an alanine (four positions before the conserved threonine in the primary sequence), which is in turn weakly hydrogen bonded to the threonine, is also present in the experimental structures of other bacterial and mammalian CYPs [45–47].
Moreover, in agreement with the DFT calculations for CYP2D6, in all these structures the conserved threonine is too far to hydrogen-bond the distal water ligand. The absence of a direct interaction between the heme axial ligand and the threonine in CYP2D6 is experimentally corroborated by the absence of differences in the electronic absorption and RR spectra of the CO adducts of the wild type and T309V, where a CO ligand occupies the position of the water molecule in the resting state. In fact, hydrogen bonding or direct electrostatic interaction between T309 and the axial CO would alter the extent of backdonation of iron dπ electrons to the empty CO π* orbitals, shifting significantly both the Fe–CO stretching frequency in RR spectra and the absorption maximum in the UV–vis absorption spectra of the reduced CO-bound heme [48, 49]. A similar situation is observed for the T268A mutant of CYP102 A1, whose CO adduct has a UV–vis absorption spectrum identical to that of the wild type [9, 14]. The absence of a significant interaction between the CO adduct and residue T309 is supported by DFT calculations on CO-bound CYP2D6, showing that the Fe–CO bond energy, bond order, and the extent of Fe → CO backdonation are virtually the same for both the wild type and the mutant (Table 1).
Besides the predominant 6cLS state, the 5cHS species is present as a minor component in RR spectra of CYP2D6 (Fig. 4). The occurrence of the 5cHS species could not be detected in UV–vis absorption spectra, probably because of its small amount. RR spectra have more, narrower bands which are usually better resolved than UV–vis spectra at room temperature, making it easier to detect asymmetries or weak shoulders when using spectral subtraction or fitting. The presence of the 5cHS species in the resting state is not unusual and it has been reported in RR spectra of other bacterial and mammalian CYPs [50–53]. A shift of the spin equilibrium toward the 5cHS state, in which the heme is no longer ligated to an axial water molecule, is often observed when a substrate (like DX for CYP2D6; Fig. 5) is present in the heme pocket, perturbing the iron-bound water molecule directly, or through inducing conformational changes in the enzyme [44]. However, this substrate-induced spin shift often does not lead to a pure 5cHS species, but rather to a mixture of the two species, with 6cLS still significantly present, like in the case of CYP2D6 with DX.
The decrease of 5cHS and the concomitant increase of the 6cLS content in the RR spectra of the T309V mutant of CYP2D6 indicate that the residue T309 is structurally involved in the spin equilibrium. This role is preserved in the presence of DX as the substrate itself perturbs the wild-type and mutant spin equilibria to the same extent, which is evident from the spectral subtraction of the ν3 region of the resting state from the substrate-bound state (Fig. 5). The same conclusion can be drawn from RR spectra of the CO adduct of the two enzymes in the presence of substrate. Moreover, the similar affinity of the two enzymes for DX (as was judged from optical titration experiments [6] as well as modeling studies [54]) does not indicate T309 to be involved in substrate binding in CYP2D6. This strongly suggests that the difference in the 6cLS-to-5cHS ratio in the RR spectra between the substrate-bound wild-type and mutant enzyme is not due to different binding modes of DX, but rather to a difference caused by the T309V mutation, as observed in the absence of a substrate.
An altered spin equilibrium upon mutation of the conserved threonine residue has been reported for many other CYPs [12, 14–17, 55, 56]. Notably in the T268A mutant of CYP102 A1, which share with the optimized CYP2D6 computational model the hydrogen-bonding pattern “distal water–alanine–threonine,” the alteration is in favor of the 6cLS, like in CYP2D6 [12, 14].
In an effort to rationalize the experimental observations, we looked for an explanatory model, compatible with our spectroscopic data, which could be tested using available quantum-mechanical methods. The absence of direct interactions between T309 and the axial ligand indicates that T309 exerts its influence on the spin equilibria of CYP2D6 indirectly.
It might be argued that the increase of the 6cLS species in the T309V mutant is due to an increased solvent accessibility of the binding pocket caused by a mutation-induced conformational change. However, this hypothesis is incompatible with the spectroscopic data on the CO adduct, which rule out any significant change in polarity in the heme pocket due to the T309V mutation. A more plausible explanation is that T309 in CYP2D6 stabilizes, by means of hydrogen bonding, an alternative position of the water that is distant enough from the iron center to yield a 5cHS state. This model has also been proposed to account for spin equilibria in substrate-bound CYP102 A1 and its T268A mutant [13, 14]. Substitution of the threonine with a valine should eliminate the hydrogen-bonding interaction and perturb the equilibrium by destabilizing the 5cHS state in favor of the 6cLS species, as observed from spectroscopic data.
DFT calculations clearly support this hypothesis, showing the destabilization of the 5cHS state upon the T309V mutation. In agreement with the experimental observation of a small amount of 5cHS in the resting state of the wild type, a spin transition from LS to HS occurs upon moving the water molecule from position 1 to 2, and a second energy minimum has been found for the water molecule hydrogen-bonded to T309 (Figs. 1c, 7). Replacing the threonine with a valine results in a relative destabilization of the structure in which water is in position 2, most likely because of the absence of hydrophilic interactions between the water and the valine. This relative destabilization of the 5c heme with the water in position 2 will result in the occurrence of a higher relative amount of the 6cLS heme species in the T309V mutant of CYP2D6 with respect to the wild type.
Although the computational model of the CYP2D6 active site used in the present study only includes a few amino acids and the iron–porphyrin complex, and therefore does not take into account the rest of the protein, it provides a clear rationale for the observed experimental data. Unfortunately, an evident limit of this model is the inability to include the substrate and to study its influence on the spin equilibrium of the substrate-bound enzymes, for which extended models and methods have to be used. However, it should be stressed that experimental data indicate that the role of T309 in the CYP2D6 spin equilibrium is independent of that of DX; therefore, it is reasonable to assume that the implications of our model for the role of T309 would still be valid for the substrate-bound enzymes.
Conclusions
On the basis of RR data, it is concluded that the T309V mutant of CYP2D6 has an altered spin equilibrium with respect to the wild-type enzyme, with a relative higher amount of 6cLS species at the expense of the 5cHS species, in both the resting state and the substrate-bound forms. Apparently, there is no direct interaction between residue T309 and the heme sixth ligand, suggesting an indirect mechanism of action on the spin equilibrium. Spectroscopic data also indicate that the T309V mutation does not significantly alter the polarity of the heme environment, excluding an increased number of water molecules in the heme pocket as the reason for the altered spin state. DFT calculations show that a simple model, involving a water molecule alternatively occupying two positions inside the heme pocket corresponding to two different spin states, is able to explain the experimental data. In this model, the position corresponding to the 5cHS state is stabilized by a hydrogen bond with T309, and a T309V mutation will induce an increase of the 6cLS species, as experimentally observed.
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t309v
cytochrome p450 2d6
resonance raman
spin equilibrium
threonine mutant